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 Macrostomum sp.

Received: 20 January 2004 / Accepted: 15 March 2004 / Published online: 9 April 2004 Springer-Verlag 2004

Abstract Macrostomid represent a group of zonula. Terminal web and zonula adherens are particu- basal bilaterians with primitive developmental and mor- larly well observed in the epidermis. During stage 6, the phological characteristics. The species Macrostomum sp., somatic primordium extends around the surface dorsally raised under laboratory conditions, has a short generation and ventrally to form a complete body wall. Muscle time of about 2–3 weeks and produces a large number of precursors extend myofilaments that are organized into a eggs year round. Using live observation, histology, elec- highly regular orthogonal network of circular, diagonal tron microscopy and immunohistochemistry we have and longitudinal fibers. Neurons of the brain primordium carried out a developmental analysis of Macrostomum sp. differentiate a commissural neuropile that extends a sin- Cleavage (stages 1–2) of this species follows a modified gle pair of ventro-lateral nerve trunks (the main longi- spiral pattern and results in a solid embryonic primordium tudinal cords) posteriorly. The primordial pharynx lu- surrounded by an external yolk layer. During stage 3, cells men fuses with the ventral epidermis anteriorly and the at the anterior and lateral periphery of the embryo evolve gut posteriorly, thereby generating a continuous digestive into the somatic primordium which gives rise to the body tract. The embryo adopts its final shape during stages 7 wall and nervous system. Cells in the center form the and 8, characterized by the morphallactic lengthening of large yolk-rich gut primordium. During stage 4, the brain the body into a U-shaped form and the condensation of primordium and the pharynx primordium appear as sym- the nervous system. metric densities anterior-ventrally within the somatic pri- mordium. Organ differentiation commences during stage Keywords Platyhelminth · Embryo · Morphogenesis · 5 when the neurons of the brain primordium extend axons Organogenesis · Differentiation 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 Introduction primordium, in the protonephridial system and, slightly later, in the lumen of the gut. Ultrastructurally, these Flatworms represent a large and diverse taxon of simple differentiating cells show the hallmarks of platyhelminth invertebrate . Recently they have again attracted epithelia, with a pronounced apical assembly of micro- the attention of developmental biologists because, in re- filaments (terminal web) inserting at the zonula adherens, gard to numerous morphological criteria, all or at least and a wide band of septate junctions underneath the some of their taxa may have branched off the phyloge- netic tree near the root of the bilaterian stock (Rieger et al. 1991; Ax 1996; Tyler 2001; Jondelius et al. 2002). Edited by J. Campos-Ortega This makes them a highly relevant system in which to J. Morris · R. Nallur · V. Hartenstein ()) study basic bilaterian developmental processes, such as Department of Molecular, Cell and Developmental Biology, establishment of the body axes and organogenesis (e.g., University of California, Hartenstein and Ehlers 2000; Younossi-Hartenstein and Los Angeles, CA, 90095, USA Hartenstein 2000a, b; Hartenstein and Jones 2003). Flat- e-mail: [email protected] worms share a number of fundamental characters with Tel.: +1-310-2067523 Fax: +1-310-2063987 coelenterates, which evolved before the bilaterians. Most notable among these characters is a single gut opening P. Ladurner · B. Egger · R. Rieger that serves as both mouth and anus, and a ciliated epi- Institute of Zoology and Limnology, dermis used for locomotion (for recent comparative de- University of Innsbruck, scription of adult flatworm anatomy, see Ehlers 1985; Technikerstrasse 25, 6020 Innsbruck, Austria 221 Rieger et al. 1991; Ax 1996). Like higher animals, most simple pharynx shared by all macrostomids places this flatworms have a subepidermal muscular layer of circu- taxon near the base of the rhabditophoran platyhelminths lar, diagonal and longitudinal fibers (see Rieger et al. (see Doe 1981 and references cited above), a classifica- 1991, 1994; Hooge 2001), and a central nervous system tion that is supported by recent molecular data (Bagu et that comprises an anterior brain and a number of longi- al. 2001; Curini-Galletti 2001; Jondelius et al. 2001; tudinal nerve cords (Rieger et al. 1991; Reuter and Littlewood et al. 2001). Macrostomid development has Halton 2001). However, the central nervous system lacks been studied several times, but only very cursorily: his- compactness, being also penetrated by muscle and gland tological and much less in vivo by Seilern-Aspang (1957) cells. Nerve tracts issuing forth from the brain extend in M. appendiculatum, in vivo by Reisinger (1923) in M. dorsally, laterally and ventrally. These tracts are con- viride, by Papi (1953) in M. appendiculatum, by Bogo- nected to up to three subepidermal nerve plexus. The molow (1949, 1960) in M. viride and M. rossicum and connectivity between neurons and muscles in flatworms finally by Ax and Borkott (1968a, b) in M. romanicum, is different from higher animals, in that, frequently, long who provided the first film material about the embryonic muscle processes (sarco-neuronal processes) approach development, but still left open a number of important the nerve fibers, instead of the other way around (see questions. The formation of the body wall was addressed Rieger et al. 1991, similar to the well studied situation in in several more recent light and electron microscopic as nematodes). The internal structure of flatworms is usu- well as histochemical studies (Tyler 1981; Reiter et al. ally referred to as being very simple. Flatworms do not 1996; E. Robatscher and B. Egger, Innsbruck, unpub- possess a coelom. The interior of the body is filled with lished results). These studies show that Macrostomum sp. the gut tube and reproductive organs, with complex as well as other Macrostomum species of the M. hys- muscle and connective tissue cells. A specialized vas- tricinum species clade (see Rieger 1977) share with other cular system or respiratory system is absent in free-living primitive flatworm groups a spiral pattern of cleavage. platyhelminths. The only tubular structures are com- However, development starts to deviate after the first prised of protonephridia (see Rohde 2001). three to four rounds of divisions from the typical spiralian Embryonic development has been studied in a number mode, in agreement with the observations by Seilern- of different flatworm species, studying live embryos and Aspang (1957). Thus, blastomeres at the vegetal pole using classical techniques of histology and electron mi- seem to spread out at the surface of the embryo and form croscopy, as well as, to a very limited extent, cell type- an outer yolk mantle (Tyler 1981). This process was specific markers, including markers for muscle and nerve called “inverse epiboly” by Thomas (1986). The transi- cells (Thomas 1986; Bagu and Boyer 1990; Reiter et tion from this stage to the final generation of body wall al. 1996; Boyer et al. 1996; Ladurner and Rieger 2000; and internal organs has not been followed in any detail Younossi-Hartenstein et al. 2000; Younossi-Hartenstein satisfactorily. In this paper, we have undertaken a de- and Hartenstein 2000a, b, 2001; Hartenstein and Jones velopmental analysis of Macrostomum sp. on the basis of 2003). Molecular tools, both as markers and as agents to observation of live embryos, histological staining of disrupt function, have been developed to study regener- sectioned and whole-mount material, electron microscopy ation and cell differentiation in , members of and immunohistochemistry. We define a series of mor- the flatworm clade Tricladida (Sanchez-Alvarado and phological stages that are in accordance with a system Newmark 1999; Sanchez-Alvarado et al. 2002; Pineda introduced for other platyhelminth taxa during recent et al. 2000, 2002; Salo et al. 2002; Cebria et al. 2002; years (Younossi-Hartenstein et al. 2000). Our work is Ogawa et al. 2002). In order to initiate a molecular aimed at providing a guide for the molecular-genetic analysis of embryonic development we have chosen the analysis of platyhelminth embryogenesis, in particular the microturbellarian Macrostomum sp. as a species that can interpretation of gene expression data from in situ hy- be easily raised in the laboratory, produces a multitude of bridization studies which are currently underway (V. eggs year round, and has a very short generation time of Hartenstein, UCLA, and P. Ladurner, Innsbruck, unpub- approximately 2–3 weeks. Macrostomum sp. is a new lished data). 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 Macrosto- Materials and methods mida within the . Based on morpholog- ical and molecular phylogenetic analysis, the latter oc- Animals cupy a position near the root of the rhabditophoran flat- Macrostomum sp. is a new marine flatworm from the Mediter- worm tree, which includes the majority of free-living and ranean, which is presently being described by Ladurner et al. parasitic taxa (Ehlers 1985; Rieger 2001; Tyler 2001; (2004). It lives in laboratory cultures of the diatom Nitzschia Littlewood and Olson 2001). Thus, macrostomids pro- curvilineata. Petri dishes with the artificial growth medium “F2” duce yolk-rich egg cells (archoophoran mode of devel- (Guillard and Ryther 1962) are inoculated with Nitzschia. By 10– 14 days the algae form a dense lawn at the bottom of the dish. opment), rather than supplying yolk to the egg by means Groups of adult Macrostomum (approximately 1.5 mm in length) of specialized yolk cells surrounding a small oocyte are transferred onto the algal dish. They produce eggs continuously, (neoophoran mode of development in all other rhabdi- and the eggs, if left undisturbed, will develop into sexually mature tophoran flatworms except polyclads). Furthermore, the adults in approximately 2–3 weeks. For preparation, eggs are col- 222 lected and fixed with 4% formaldehyde in PBS buffer. They are Results 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 Overview and staging system same protocols that have been established for Drosophila embryos (Ashburner 1989). Embryonic development of Macrostomum sp. takes ap- proximately 120 h at 20C. We have subdivided this Electron microscopy and histology phase into eight stages, which can be distinguished on the basis of several morphological criteria in living material, Timed stages of embryos of Macrostomum sp. were punctured with as well as fixed and sectioned preparations. The definition 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).

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 fix- ation 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 Fig. 1 Overview of stages of Macrostomum sp. development. (1:1). They were then mounted in a drop of fresh Epon and cov- Panels show highly schematic drawings of embryos of increasing erslipped. Representative specimens were embedded in Epon and age, shown in lateral view, illustrating the major events of Mac- sectioned (1–3 mm) on an LKB ultramicrotome. Sections were rostomum sp. embryogenesis. Anterior is to the left and dorsal to counterstained with methylene blue/toluidine blue/borax (Ash- the top. The number at the top left of each drawing indicates the burner 1989). Preparations were analyzed and photographed with a embryonic stage as defined in the text. Tissues are shaded in dif- Zeiss Axiophot photomicroscope and a Biorad MRC1024ES mi- ferent colors as shown at the bottom left of the panel. For details see croscope using Laser sharp version 3.2 software. Figures were as- text (br brain primordium, cx cortex, emp embryonic primordium, sembled and lettered with Adobe Photoshop 6.02 (Adobe). 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 primordi- um, 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: cleav- Fig. 3a–d Early stages of Macrostomum sp. development: epiboly age (stage 1). a Photograph of living embryo at four-cell stage. b, c of hull cells (stage 2). a Photograph of living embryo. b Whole- Photographs of embryo at eight-cell stage, view from pole; mount of embryo stained with basic fuchsin in ventral view. c, d focal plane through micromere quartet 1a–d (b) and macromere Cross sections near anterior and posterior pole, respectively. Hull quartet (c). Note larger size of 1D macromere and relatively large cells have extended over the surface of the embryo and form the size of micromeres. d Parasagittal section showing yolk-rich animal external yolk mantle (eym). Proliferating blastomeres enclosed by blastomeres (bl) and incipient hull cells (hc; pb polar bodies; scale the external yolk mantle represent the embryonic primordium (emp bar 20 mm) in a). Within this mesenchymal cell mass, small cells are clustered near one pole, and larger, yolk-rich cells near the other. We pre- sume that the smaller cells will give rise to the body wall and of these embryonic stages is largely based on a similar nervous system (somatic primordium; sop in b and c), and the large-sized cells will form the gut (gut primordium, gp). Arrow in b system that was developed for the rhabdocoel flatworm points at one of numerous mitotic figures that occur throughout Mesostoma lingua (Younossi-Hartenstein et al. 2000), and early development in somatic primordium and gut primordium (nhc has since then been adapted for other archoophoran and hull cell nucleus; scale bar 20 mm) neoophoran flatworm species (Younossi-Hartenstein and Hartenstein 2000a, b, 2001; Hartenstein and Ehlers 2000; Ramachandra et al. 2002). In the following, we will place the yolk and form the definitive epidermis. A neu- briefly introduce and define the characteristic features of ropile forms in the center of the brain primordium; the eight stages (Fig. 1). pharynx and gut become lined by ciliated cells. During Stage 1 (0–15 h) represents early cleavage, during stage 6, the formation of the body wall is completed. The which the zygote divides basically in a spiral pattern. In epidermis spreads around the embryo. Gland cells con- vivo cleavage can only be observed until the third cleav- taining rhabdites are scattered throughout the epidermis. age, after which the opaque yolk granules obscure the Myoblasts form a regular, orthogonal grid of fibers un- borders of individual blastomeres. During stage 2 (15– derneath the epidermis. During stage 7, the previously 30 h), a small number of yolk-rich, presumably vegetal spherical shape of the embryo transforms to an elongated blastomeres with large nuclei expand and surround the shape and the embryo bends its caudal end towards the other blastomeres (yolk mantle), which form a prolifer- ventral side of the anterior body half. The brain primor- ating mass (here referred to as the embryonic primordi- dium condenses, and pigmented eyespots form in the um) in the center of the embryo. Stage 3 (30–45 h) is dorsal brain cortex. Stage 8, comprising the final 10–15 h characterized by the expansion and diversification of the of embryonic development, resembles the fully elongated embryonic primordium. Anteriorly and laterally, cells of and differentiated freshly hatched juvenile. 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. Early embryogenesis: cleavage and formation During stage 4 (45–60 h), the outer yolk mantle has be- of the embryonic primordium (stages 1–3) come very thin. Definitive primordia of the brain and pharynx can be distinguished at the anterior pole. Stage 5 Our data confirm previous observations (Reisinger 1923; (60–75 h) is defined by the onset of tissue and organ Bogomolow 1949, 1960; Papi 1953; Seilern-Aspang 1957; differentiation. Outer cells along the lateral margins dis- Ax and Borkott 1968a, b) stating that early cleavage 224

Fig. 4a–d Early organogenesis in Macrostomum sp.: emergence of Fig. 5a–d Organogenesis of Macrostomum sp.: onset of organ organ primordia (stage 4). a Photograph of living embryo. b differentiation (stage 5). a Photograph of living embryo, anterior Whole-mount of fuchsin-labeled embryo in dorsal view. c Cross view. b Tilted cross sections of embryo near anterior pole. c, d sections of somatic primordium near anterior pole. d Cross section Whole-mounts of fuchsin-labeled embryos in ventral view (c) and at mid-levels of embryo. At the anterior of the embryo the pri- lateral view (d). Cells at the outer surface of the somatic primor- mordium of the brain (br) appears as a bilateral condensation within dium have undergone a mesenchymal-epithelial transition and form the somatic primordium (sop). The somatic primordium forms a the primordium of the epidermis (ep). Deep cells of the somatic belt around the equator of the embryo. It measures 20–25 cell primordium form precursors of muscle cells (sm; arrowheads in b) diameters in length (AP axis), 10–15 cell diameters in width (DV and other cell types associated with the body wall. The somatic axis) and three to four cell diameters in thickness, bringing the primordium does not yet form a complete body wall; dorsally and overall cell number up to 2–3,000. Most cells are post-mitotic, ventrally, remnants of the external yolk mantle (eym) still abut the evidenced by the scarcity of mitotic figures from stage 4 onwards, surface. Neurons of the brain primordium have differentiated and as well as the prevalence of small, dense nuclei. The somatic pri- form a central neuropile (np) surrounded by a cortex (cx) of somata. mordium is still covered by a thin yolk mantle (eym in c). The gut Posterior to the brain is the primordium of the pharynx (ph), formed primordium (gp) lies in the center of the embryo and contains large, by a cylindrical array of columnar epithelial cells. Arrow in (d) yolk-rich cells (nhc hull cell nucleus; scale bar 20 mm) points at cells that close the pharynx lumen ventrally. Note the folding of the eggshell ventral of the arrow, which probably con- stitutes an artefact related to shrinkage of the preparation. Precur- follows a pattern that shows characteristics of quartet- sors of muscle cells around the pharynx are indistinct, small, spiral cleavage (Fig. 2). Some significant deviations from spindle-shaped cells. The gut primordium (gp) contains large, yolk- spiral cleavage, as seen for instance in polyclads, do oc- rich masses, interspersed with small and dense yolk spheres (ys). The posterior of the embryo contains the primordium of the tail cur. Micromeres are not substantially smaller than mac- plate (tp). Scale bars 20 mm romeres (Fig. 2b). At the stage when more than one mi- cromere 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 Fig. 6a–j Organogenesis in Macrostomum sp.: ultrastructure of organ primordia. a–j Electron micrographs of details of cross (Dottermantel) that gradually becomes thinner as devel- sections of a stage 5 embryo. a Overview of somatic primordium, opment progresses (Figs. 1, 3b, d, 4c, d, 5d). The indi- containing superficial layer of epidermal precursors (ep) and deep vidual hull cells are filled with yolk granules and show layer of muscle precursors and neural precursors (ne). Epidermal elongated nuclei which are larger than those of most precursors are cylindrical, ciliated (ci) epithelial cells. Numerous yolk granules (yg) are still present in the basal portion of the epi- blastomeres (Fig. 2d). dermal precursors. b Border between epidermal primordium (ep) Once internalized, the cleaving embryonic primordium and external yolk mantle (eym). c Magnified views of part of b, forms a mass surrounded by yolk-rich hull cells (stage 2; showing details of the junctional complex formed between epi- Fig. 3). The time course of mitotic divisions and spindle dermal precursors. The complex is formed by a pronounced, septate junction (sj) near the apical pole of the cell. Further apical still is orientation appears to be quite irregular, and cannot be the zonula adherens (aj) with its characteristic membrane density followed without special markers or dye injections. Cells and cytoplasmic plaque. An unusual feature also noticed at later are variable in size and contain yolk granules to a greater 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 epidermal cell, showing cilia (ci), zonula adherens (aj), septate the junctional complex formed between the epidermal precursor junction (sj), and terminal web (tw; microfilaments inserting at the (ep) and external yolk mantle (eym). The septate junction is similar zonula adherens). Apical of the terminal web is a layer of ultra- to the one between epidermal cells; the adherens junction is not as rhabdites (urh), which are small secretory granules of epidermal wide and lacks a cytoplasmic plaque on the side of the yolk mantle cells. Mitochondria (mit) are clustered underneath the terminal (arrowhead). e, f Magnified views of part of a, showing details of web. h, I Detail of cell that does not yet form part of the epidermal bundles of cell processes (cp), probably axons, located at the primordium but has started formation of cilia (ci). Scale bars:2mm border between neural precursors (ne) and epidermal cells (ep). a;1mm b, e, g, h Myofilaments are not yet formed at this stage. g, j Apical part of 226 or lesser extent. Anterior cells, which generally seem of cell bodies separates the neuropile from the developing smaller in size than posterior cells (Fig. 3b, c), constitute pharynx (Fig. 5b). At the beginning, the brain primordi- the precursors of epidermis, body-wall muscles and ner- um is wide (in the transverse axis) and flat (in the AP vous system (somatic primordium). During stage 3, the axis); as development progresses, the primordium con- somatic primordium expands posteriorly, forming two denses, becoming increasingly narrower and thicker (com- symmetric plates along the lateral sides of the embryo. It pare Figs. 5b and 11b). is unclear whether this expansion involves actual cell The pharynx primordium appears postero-ventrally of migration, or whether cells at posterior levels which ear- the brain primordium as a cylindrical array of cuboidal lier had been large and yolk-rich, decrease in size and cells that develop as the ciliated pharynx epithelium then join the somatic primordium. Blastomeres in the (Fig. 5b–d, see also later stages, e.g. Figs. 7d, 10f). As has center of the embryo (gut primordium) remain large and been described for other flatworm species (see Thomas yolk-rich (Fig. 3d). These cells later transform into the gut 1986 and Bagu and Boyer 1990 for summaries), the epithelium (Fig. 4d). 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 The formation of organ primordia (stages 4 and 5) initially seems to have no connection to the epidermis, or to the gut lumen. The connection forms during stage 6 By mid-embryogenesis (stage 4) proliferation becomes when the pharynx primordium elongates both dorsally less pronounced and organ primordia take shape. The and ventrally, and establishes contact with the ventral most prominent primordium is that of the brain, forming a epidermis. bilaterally symmetric mass of densely packed cells at the Muscle precursors initially appear as elongated and anterior pole of the embryo (Fig. 4a, b). Posterior to the flattened cells forming irregular layers underneath the brain, the somatic primordium consists of intermingled epidermal primordium (see also Reiter et al. 1996 for M. epidermal, gland, muscle and other precursors that form hystricinum). Muscle precursors are concentrated in a plates alongside the lateral surface of the embryo (Fig. 4a, bilateral band that extends along the flanks of the embryo b). The external yolk mantle still covers the surface, but is (Figs. 5b, 7b). Fewer muscle precursors populate the compressed into a thin layer (Fig. 4c, d). The gut pri- space between epidermis and brain primordium, and mordium fills the posterior-central part of the embryo around the pharynx. Muscle differentiation sets in towards (Fig. 4a, b, d). Starting towards the end of stage 4, pe- the end of stage 5 with the formation of myofilament- culiar spherical structures can be distinguished within containing processes which form a highly regular network the gut primordium (Figs. 4d, 5d, 7e, 10f). These “yolk of longitudinal, circular and diagonal fibers (see section spheres” stand out in histological sections, in electron Muscular system). micrographs (not shown) and can even be seen in living A conspicuous condensation of cells in the deep layers material (not shown). of the somatic primordium at the posterior tip of the The appearance of definitive organ primordia occurs embryo demarcates the primordium of the tail plate concomitantly with the onset of cellular differentiation (Fig. 5c) The tail plate primordium consists of muscles during stage 5. Most prominent during this stage are the and glands of the duo-gland adhesive system (Tyler epidermis, brain, pharynx and gut. Cells located at the 1988), that allow the freshly hatched animal to quickly external surface of the embryonic primordium transform grab on to the substrate. Cells from this caudal region into a ciliated epithelial epidermal layer that displaces the later may also give rise to the external genitalia, i.e., the external yolk mantle (Figs. 5, 6a–d). The onset of ciliation genital pores and the male copulatory organ. Along with of the multiciliated cells has been described in detail by the gonads, the external genitalia apparently do not dif- Tyler (1981). As of yet we do not have data that indicate ferentiate during the embryonic period, and will not be the resorption of the yolk mantle by the primary epider- considered here. mis. 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 Organ differentiation (stages 6–8) development proceeds, the epidermis and underlying cells of the somatic primordium stretch in the transverse axis Final organ formation takes place during the last 2 days of and fully enclose the embryo by the end of stage 6. the 5-day embryonic period (stages 6–8; Figs. 7, 8, 9, 10, Cells located underneath the epidermis differentiate 11, 12, 13). 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 primor- Epidermis dium, resulting in the formation of a typical inverte- brate ganglion, which consists of a cortex of neuronal cell During early stages (5 and 6) the epidermal primordium bodies surrounding a central neuropile (Fig. 5b, c, f; see consists of cylindrical multiciliated epithelial cells also Rieger 1998). The cortex is three to four cell bodies (Fig. 6a, b). Underneath the apical membrane the terminal in thickness, except posteriorly where only a single layer 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-la- beled 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 ex- ternal 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), phar- ynx (ph) and gut (gp) can be clearly distinguished in the liv- ing embryo. In fixed whole- mounts (b, c), the brain and pharynx primordia stand out. Nuclei of gut cells are not la- beled optimally. The increasing number of axons leads to a thickening of the neuropile (np). Underneath the epidermis, so- matic muscle precursors (sm in b, c, g) have differentiated and form flat, elongated cells that produce a regular network of circular, diagonal and longitu- dinal fibers. The pharynx has lengthened (c) and cilia can be recognized under light micros- copy in the lumen of the phar- ynx (b, d), as well as the gut (e). The gut primordium has reor- ganized 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:20mm a–e;10mm f–h

oped (Fig. 6g, j). Ultrarhabdites (or epitheliosomes; for established by stage 5. It consists of an apical zonula terminology see Rieger et al. 1991), small vesicles above adherens and a wide belt of septate junctions below the terminal web, are already present between the termi- (Fig. 6g, j). Of special significance is that septate junc- nal web and the apical membrane (Fig. 6d, g). Basally, tions and adherens junctions also join neighboring cell epidermal cells still contain numerous yolk granules, membranes at the boundary between epidermal primor- which will decrease as development proceeds (Fig. 6a). dium and hull cells (Fig. 6c). The zonula adherens ex- The junctional complex in between epidermal cells is well hibits the characteristic membrane thickening and sub- 228

Fig. 8a–i Organogenesis in Macrostomum sp.: ultrastructure of the iated epidermal layer (ep), subepidermal muscle fibers (mf), main body wall. a–i Electron micrographs of details of longitudinal longitudinal cord (mln) and gut primordium (gp). Epidermal cells sections of a stage 6 embryo. a Overview of body wall, with cil- still contain yolk granules (yg) near their basal pole. b Magnified 229 membraneous plaque. However, the cleft that separates Brain cells at the level of the zonula adherens is wider, rather than narrower, than the intercellular cleft at more basal Brain neurons differentiate during stages 5 and 6 when the levels (Fig. 6g, see also below, Fig. 9d). This feature of brain primordium forms a broad and flat structure at the the zonula adherens (“open zonula adherens”) has also anterior tip of the animal (Figs. 5b, 6b, c). Whole-mounts been observed in embryos of other flatworm species labeled with anti-tyrosinated tubulin (tyrTub; Fig. 12) (Younossi-Hartenstein et al. 2000) and is generally ob- reveal that the brain cortex is formed by multiple clusters served in adult specimens which were anesthetized with of five to ten neurons each. These clusters (possibly lin- isotonic MgCl2 solutions (Tyler 1984). eages) extend axons that fasciculate together, forming a Later epidermal cells become cuboidal to squamous, thin tract; several such tracts in turn converge and form a except for the anterior region (apical plate) and tail plate, distinct neuropile compartment. We will describe details which maintains more cylindrical epidermal cells (Fig. 9a, of the anatomy of the juvenile and late embryonic brain d). Microvilli become very prominent. A thin and patchy elsewhere (Morris et al., in preparation) and will therefore ECM separates the epidermis from the underlying muscle keep the description of the nervous system brief. and nerve cells (Fig. 9a, h). It has been observed in other During stage 5 (Fig. 12a–c), four large systems of macrostomids (Rieger et al. 1991) that epidermal cells tracts that lay down the brain neuropile can be distin- form extensive sheath-like processes at their baso-lateral guished. Each system is formed by six to eight converging membrane, which intercalate between neighboring cells, axon fascicles; during later stages, as more neurons dif- suggesting that this interdigitation of epidermal cells may ferentiate, fascicles are added to each system. Medially in compensate for the absence of a continuous basement the brain, a dorsal and ventral medial longitudinal system membrane. However, interdigitation of epidermal cells is (MLV, MLD) is laid down. Axon fascicles contributing not a significant feature of epidermal cells in Macrosto- to these systems are formed by neuronal clusters located mum sp. (Fig. 9a). The apical junctional complex between in the anterior cortex; to a lesser extent, posterior neu- epidermal cells remains similar to the one described for rons with anteriorly projecting axons also exist. The stage 5, with a wide and prominent septate junction and an medial longitudinal systems can be followed throughout open zonula adherens (Fig. 9d). Epidermal nuclei acquire development into the brain of freshly hatched juveniles their characteristic multilobulate shape during stage 7 (Fig. 12g–I); at this stage, following brain condensation, (Fig. 13a). During this stage, rhabdite-filled gland cells systems of both sides approach each other and are sepa- with subepidermal cell bodies and long excretory ducts rated only by a narrow cleft. Adjacent to the longitudinal permeating the epidermis can be distinguished by light systems are the medio-dorsal and medio-ventral com- (Fig. 11f) and electron microscopy (Fig. 9d). These missural fiber systems (MCD, MCV). The MCD forms rhabdite glands are rather evenly distributed over the the largest system in the early embryo. It comprises dorso-lateral sides of the body (Ladurner et al. 2004). commissural axon fascicles emitted by at least ten clusters Specialized glands are the adhesive glands in the tail plate of neurons located in the dorsal cortex. Fewer axon fas- (Fig. 9j), as well as a population of rhammite glands cicles of neuronal clusters located in the ventro-medial whose cell bodies are located posterior to the brain and cortex converge to form the ventro-medial commissural whose long necks cross the brain to terminate in the apical tract (MCV; Fig. 12) Finally, axons of neuronal clusters in plate (not shown). 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. view of apical part of epidermal cell with cilia (ci), ultrarhabdites Main longitudinal nerve cords (urh), terminal web (tw), zonula adherens (aj) and septate junction and protonephridial system (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 The main longitudinal nerve cords (MLN) extend from extracellular material (basement membrane, bm) separate nerve and the posterior surface of the brain towards the tail of the muscle fibers from overlying epidermis. d, e Muscle cell body (sm), embryo (Figs. 10c, 11e, 12g). They form one large post- circular muscle fiber (cf) and longitudinal muscle fiber (lf). Note close contact between axons (ax; magnified view in e) and muscle pharyngeal commissure (not shown). Densely packed cell plexus. f Overview of sagittal section reaching from brain (to the bodies and muscle cells accompany the axons. The pro- left of panel) and pharynx (right), showing neurons (ne), neuropile tonephridial system extends parallel and dorsal to the (np), muscle fiber piercing brain cortex (brm), pharynx epithelium MLN (Figs. 9a, b, 12k, l). In the hatching juvenile, the (phe) and pharynx muscle (phm). g High magnification of central MLN (at a level posterior to the post-pharyngeal com- neuropile. h Root of main longitudinal nerve cord (mln) and at- tached muscle cells (sm) and longitudinal muscle fibers (lf). I High missure) contains approximately 100 axons. The MLN magnification of deep array of cilia (ci) surrounded by pro- reaches towards the tail where it thickens to form a caudal tonephridial precursor cell (pn). Scale bars 1 mm ganglion associated with the muscles and glands of the 230

Fig. 9a–j Ultrastructure of hatching juvenile. a–d Cross sections epithelium (ge). Epidermal cells are covered apically by microvilli of body wall, showing epidermis (ep), subepidermal gland cells (mv in d) and cilia (ci). Pronounced junctional complex (jc) in- (glc) with rhabdites (rhb), muscle fibers (mf), protonephridia (pn) terconnects neighboring cells. Microfilaments form terminal web with ciliated lumen (ci), main longitudinal nerve cord (mln) and gut (tw). e–g Cross section of posterior brain (br) and pharynx (phe). 231 tail plate (not shown). The protonephridial system is the interior of the animal where they form a muscle net formed by a pair of longitudinal tubes which gives off around the brain neuropile and the pharynx, respectively several evenly space side branches. The tube and side (Fig. 13a, b). The brain-related deep muscle plexus arises branches are lined by a flat epithelium. AcTub and tyrTub in the anterior third of the animal. It forms a group of labeling of embryo whole-mounts reveal the cilia in crescent-shaped fibers that in part skirt the outer surface protonephridia for the first time during late stage 5 of the brain, in part pass through the cortex and neuropile (Fig. 12k). Specialized cells called flame cells or cyrto- (see also Rieger et al. 1991, Fig. 12, p 144 and Rieger et cytes cap the blind ends of the side branches. Pro- al. 1994, Fig. 3c for M. hystricinum marinum). These fi- tonephridial tubules are readily detectable in electron bers seem to be invariant in relationship to other com- micrographs of late embryos (Fig. 9a, b). In cross section ponents of the brain and can serve as landmarks for they appear as electron dense cells with a 2–4 mm wide subdividing the brain into anatomically defined com- inner lumen, filled with densely packed cilia. In ventral partments (see also unvaried condition in freshly hatched view they appear as five to six regularly spaced, cylin- juvenile of M. hystricinum marinum in Rieger et al. drical structures (Fig. 12l) that are easily mistaken for 1994). axons, in particular due to the fact that they are located so The second deep muscle grid branches off the sub- close to the main nerve cord. We assume that the early epidermal grid in the mid body and is organizing the acTub labeling corresponds to the short, cyrtocyte-con- pharynx, thereby serving as the structural support (“pha- taining branches of the protonephridial system. In hatched ryngeal suspensor”) anchoring the pharynx to the body juveniles, a continuous longitudinal trunk that opens an- wall (“pharynx holding apparatus” in freshly hatched ju- teriorly into the pharynx lumen is labeled in addition to veniles, see Rieger et al. 1994 for M. hystricinum mar- the cyrtocytes (not shown). inum). Ventrally, pharyngeal suspensor fibers extend for- ward underneath the gut, then continue as longitudinal fibres on the pharynx wall (Fig. 13c, d). Dorsal suspensor Muscular system fibers pass over the dorsal gut surface, then curve ven- trally, pass between brain and pharynx, and continue as Muscle cells form a grid of extraordinary regularity un- longitudinal fibers in the ventro-anterior pharyngeal wall. derneath the epidermis; they also surround the neuropile Together with an inner layer of circular muscles, these of the brain, the pharynx, gut and male copulatory ap- longitudinal fibers form the intrinsic musculature of the paratus (see post-embryonic differentiation in Rieger et pharynx (for adults, see also Doe 1981). al. 1991, 1994 in M. hystricinum). The subepidermal Muscle differentiation is initiated during stages 5 and muscular plexus of the late embryo forms a pattern that 6. The embryonic muscle pattern has been documented is closely correlated to the pattern of epidermal cells by Reiter et al. (1996) who used phalloidin to visualize (Fig. 13). This is particularly obvious in the case of the the actin-rich myofilaments. Our materials can add little longitudinal fibers, which are thicker and more invariant to this description. Muscle precursors initially form a in number and spacing than the circular and diagonal fi- rather irregular layer underneath the epidermal primor- bers. As evident from Z-projections of a series of confocal dium (Fig. 5b, c). During stage 6, these cells elongate and sections in which both muscle fibers and epidermal nuclei send out processes that are preferentially arranged lon- are labeled, there exists an almost perfect 1:1 relationship gitudinally. Diagonal and circular fibers appear to de- between rows of epidermal nuclei and longitudinal fibers velop later. Fibers are 0.5–1 mm in diameter (Figs. 8d, h, (Fig. 13a, b). The epidermis in freshly hatched juveniles 9d, h); based on the findings of Reiter et al. (1996), measures approximately 30–35 cells in perimeter (at mid- longitudinal fibers are much fewer and extend almost body level), and possesses the same number of longitu- along the entire length of the animal, and circular fibers dinal muscle fibers. surround half of the circumference. Given the close Two systems of preferentially longitudinal fibers di- correlation between muscle fibers and epidermal cells in verge from the subepidermal muscle grid and extend into 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 Brain neuropile (np) is separated from pharynx only by a scattered tissue layers control the positioning of muscle fibers and population of neuronal cell bodies (ne). Muscle fibers (mf) traverse epidermal cells. the neuropile. Densely ciliated, cylindrical pharynx epithelium Freshly hatched juveniles possess a thin layer of mus- (phe) surrounds a circular lumen (phl). Muscle cells form a thin cles surrounding the gut (Ch. Seifert, Innsbruck, unpub- layer of scattered fibers (phm) at the basal surface of the epithelium. lished results). In preparations of embryos, gutassociated Necks of gland cells (pgl) terminate bilaterally in pharynx lumen. h Section of body wall. An elongated muscle cell (sm) gives rise to a muscles were undetectable; we therefore assume that longitudinal fiber (mf). A thin basement membrane (bm) separates formation of this visceral muscle layer must take place epidermis (ep) and muscle layer. I Cross section of gut with internal shortly before hatching. The musculature surrounding the lumen (gtl) and gut epithelial cells (ge) covered by cilia (ci). j genitalia develops post-embryonically. Section of adhesive glands (agl) clustered in the tail plate at pos- terior tip of animal. Scale bar 1 mm(panels B, C, F, G are twofold magnifications of areas outlined by rectangles in a and e, respec- tively) 232 Fig. 10a–h Late phase of or- ganogenesis (stage 7). a Photo- graph of living embryo, ventral view, b, c Whole-mount of fuchsin-labeled embryo in ven- tral view. d, e Parasagittal and horizontal histological sections of embryo. f–h Magnified de- tails of section shown in e. a Embryo has flattened and elon- gated and moves actively in the eggshell. A characteristic fea- ture of stage 7 are the pig- mented eyespots (eye) embed- ded in the brain (br). b, c Brain has condensed in medio-lateral axis (compare with corre- sponding views of stage 6 and stage 5 embryos shown in pre- vious figures). The eyes are embedded in the dorsal brain cortex (cx in b). Main longitu- dinal 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 neu- ropile (np) and cortex (cx), pharynx (ph; arrow points at junction between lumina of gut and pharynx), gut epithelium (ge), and tail plate (tp). f Mag- nified 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;10mm f–h

Pharynx and gut ganization of myoblasts that is so characteristic of the pharynx primordium of higher flatworms is absent. The During stages 5 and 6, the pharynx primordium appears as pharynx primordium starts out as a cylinder three to four a rosette-shaped structure that is closely attached to the cell diameters high and six to eight cell diameters in pe- posterior surface of the brain (Figs. 5b, c, 7b, c). Com- rimeter. These cells form the epithelial lining of the pared to other flatworms, the number of cells forming part pharynx. In the interior of the embryo, the pharyngeal of the pharynx primordium is not high, and a radial or- lumen borders the gut primordium of the early embryo; 233 Fig. 11a–f Late phase of orga- nogenesis (stage 8). a Photo- graph of living embryo, lateral view. b, c Whole-mount of fuchsin-labeled embryo in ven- tral 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 tak- en 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 pro- tonephridia (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 mus- cles, tp tail plate). Scale bars: 20 mm a, b, d–f;10mm c

ventrally, the pharynx lumen initially seems to be closed terminal web and junctional complex (Fig. 9e, g). In late off by hull cells or precursors of the epidermis (Fig. 5c, embryos, gland cells with long secretory ducts can be arrow). During stage 6, the pharynx elongates and forms seen to open into the pharynx lumen (Fig. 9g). A sparse an opening at the ventral surface (Fig. 7c, arrow). Pharynx network of circular muscle fibers, as well as neurons and epithelial cells resemble epidermal cells in their ultra- axon fascicles, surround the pharynx epithelium (Fig. 9g). structural differentiation, including the apical ciliation, 234 235 Formation of the gut is difficult to follow, because New aspects of the quartet-spiral development cell boundaries are obscured by the high yolk content of in platyhelminthes and the evolution of ectolecithal eggs gut precursors. From late stage 6 onward a narrow cen- of the tral lumen surrounded by cilia can be observed (Fig. 7e). Initially, the gut lumen is closed on all sides (Fig. 12k); Macrostomid development begins with a spiral cleavage during stage 7, the inner end of the pharynx connects to of the entolecithal egg, a feature it shares with all other the gut, and the lumina of pharynx and gut become con- archoophoran “turbellarians”, including polyclads and fluent (Fig. 10c, e, arrow). The gut epithelial cells that acoels. Subsequently, during later cleavage (stage 2) the surround the lumen and carry cilia on their apical surface developmental path of Macrostomum sp. starts to deviate are few in number and reach a large size, compared to from that of typical spiralian embryos (as represented by other cells of the late embryo. During intermediate stages the polyclads), in that some blastomeres extend around of gut development (stages 4–6), cell borders are diffi- the embryonic primordium (a process called “inverse cult to discern light microscopically. Whether the gut epiboly” by Thomas 1986) and give rise to a layer of primordium represents a true dynamic syncytium will yolk-rich hull cells that form an external yolk mantle (see remain an interesting problem for future studies. Tyler 1981, for the M. hystricinum species group defined by Rieger 1977; Gehlen and Lochs 1990). In a Macros- tomum species, such a yolk mantle originating from hull Discussion cells was first reported by Seilern-Aspang (1957 for M. appendiculatum) who described the formation of these In this paper, we have described the principal steps in cells from early vegetal blastomeres. For a similar spe- morphogenesis that shape the embryonic development of cies, Papi (1953) remarks that the fate of blastomeres the macrostomid flatworm, Macrostomum sp. We have could not be followed in vivo beyond the eight-cell stage. employed a morphological staging system recently in- In other Macrostomum species hull cells have not been troduced for the rhabdocoel species Mesostoma lingua mentioned, but may have simply been overlooked (see (Hartenstein and Ehlers 2000), and thereafter adapted Reisinger 1923, for M. viride). Blastomeres could be to other flatworm taxa, including polyclads (Younossi- followed during a typical quartet-spiral cleavage up to the Hartenstein and Hartenstein 2000b) and acoels (Rama- 64-cell stage by Bogomolow (1949, 1960) for M. viride chandra et al. 2002). The stages facilitate the comparison and M. rossicum). Ax and Borkott (1968a, b, for M. ro- of developmental events in different groups, and will add manicum) refer to a hull membrane that obscures the a useful tool to the description of gene expression patterns embryo after the 16-cell stage. that form part of the molecular-genetic analysis of em- Based on stylet morphology, all species of the genus bryogenesis currently under way. 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 Mac- rostomum species described by Bogomolow (1949, 1960) Fig. 12a–l Organogenesis in Macrostomum sp.: development of the warrants further investigation. nervous system and protonephridia. a–l Confocal sections of whole- Gastrulation in Macrostomum sp. also deviates sig- mounts of embryos at stage 5 (a–c), stage 6 (d–f) and stage 8 (g–h) nificantly from the typical spiralian mode that prevails in labeled with tyrTub antibody, which labels microtubular skeleton. other archoophorans. Previous studies had not provided 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 inter- any details on gastrulation because, as already stated by mediate level, and lower row (c, f, I) at ventral level of developing Seilern-Aspang (1957) for M. appendiculatum, the mode neuropile. Labeling shows outline of neuronal cell bodies and axons. and type of gastrulation in living embryos cannot be ob- At stage 5 (a–c), several neuropile founder clusters can be distin- served due to the presence of the yolk mantle. Our data guished: dorsal medial longitudinal cluster (mld), ventral medial suggest a morphogenetic process that leaves out typical longitudinal cluster (mlv), dorsal medial commissural cluster (mcd), ventral commissural cluster (mcv), and lateral commissural cluster gastrulation movements and, like the formation of the (lc). Axon bundles formed by these clusters are later joined by more external yolk mantle, bears resemblance to early devel- fibers, resulting in the growth and compaction of the neuropile. opmental stages in neoophorans (Bresslau 1904; Harten- Axons forming the main longitudinal cord (mln) that extends into stein and Ehlers 2000). Thus, following the establishment the trunk can be distinguished from stage 6 onward (not shown) and are prominent at late stages (g). TyrTub antibody also labels cilia of of the yolk mantle, cells in the interior of the embryo sort epidermis (ep), pharynx (ph) and protonephridia (pn). j, k Whole- out into a yolk-rich gut primordium, formed by large cells mount of stage 5 embryo labeled with acTub antibody labeling cilia located more centrally, and a somatic primordium that of epidermis (j), pharynx (k; ph), gut lumen (gtl) and protonephridia consists of smaller cells located anteriorly. These cells (pn). l Whole-mount of stage 7 embryo labeled with acTub. Pro- tonephridia (pn) are formed by tubules arranged in a row that ex- gradually expand posteriorly and form plate-like mesen- tends on either side of the embryo dorsal of the main longitudinal chymal cell masses on either side of the gut primordium. nerve cord. Scale bars:10mm a–l;20mm j–l 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-pro- jection of five consecutive confocal sections (1-mm inter- val) showing pattern of epider- mal nuclei (ep) and somatic muscle fibers (sm) of ventral body wall. b Magnified view illustrating one-to-one relation- ship between columns of epi- dermal nuclei and longitudinal muscle fibers. c Z-projection of 12 consecutive confocal sec- tions (1-mm interval) showing deep systems of muscle fibers, consisting of brain-related muscles (brm), pharyngeal sus- pensors (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 phar- ynx, vm visceral muscle; scale bar 20 mm)

of cell differentiation, does the somatic primordium ex- yolk cells (“Vitellocytenepithel”). Later, a secondary set pand dorsally and ventrally to enclose the gut primordi- of hull cells that arise from blastomeres displace the um. The difference between this peculiar mode of body vitellocytes. Such blastomere-derived hull cells have also wall formation and the corresponding process in poly- been described for other proseriates (Minona, Bothrio- clads and other spiralians is evident. Thus, in polyclads, plana, Otomesostoma) as well as for the lecithoepitheliate hull cells are absent. The definitive epidermis primordium Xenoprorhynchus by Reisinger et al. (1974a, b). is formed at an early stage during gastrulation by animal The formation of an external yolk mantle by hull cells micromeres, which by epibolic movements stretch over and the absence of a characteristic gastrulation are two the large, yolk-rich cells at the vegetal pole (Surface criteria which would place macrostomids between the 1908; Boyer et al. 1996, 1998; Younossi-Hartenstein and more “basal” archoophorans (e.g., polyclads) and neoo- Hartenstein 2000b). The latter include the descendants of phorans. This is in contrast to other criteria, such as the the fourth quartet micromere that form the mesoderm and structure of the pharynx, which, in macrostomids, is of the the gut, and the macromeres providing yolk that will pharynx simplex type, whereas poIyclads show the more come to be located in the lumen of the gut, or (in case of advanced pharynx plicatus type. More cytological studies polyclad flatworms) stay rather small and degenerate at on the hull cells are needed. In any event, the new data on an early stage (Surface 1908). the embryonic development of Macrostomum presented The origin of hull cells from blastomeres may have here open new avenues to investigate the origin of the evolutionary significance for the development of ecto- heterocellular gonad with ectolecithal eggs and highly lecithal eggs, an issue that clearly needs further investi- derived embryonic development from the basic quartet- gation. Ax (1961) drew attention to the different onto- spiral cleavage of primitive archoophorans. genetic origin of hull cells in certain archoophora and The inverse epiboly seen in Macrostomum sp. (of the neoophora. In the latter, hull cells have been described as M. tuba species group) and certain other macrostomid descendants of yolk cells (vitellocytes; Bresslau 1904; species (of the M. hystricinum species group) poses an Thomas 1986; Hartenstein and Ehlers 2000). Giesa (1966) interesting phenomenon also from a developmental per- also describes, for the proseriate Monocelis, a primary spective. Spiralian cleavage is commonly seen as a de- cover of the ectolecithal embryo formed by specialized velopmental mechanism that generates fixed lineages. 237 Injecting early blastomeres with lineage tracers has re- pharynx primordium appears directly posterior of the brain vealed a high degree of invariance in regard to what or- (Younossi-Hartenstein and Hartenstein 2000a, 2001); in gans and cell types of the hatching larva are derived from the typhloplanoid (rhabdocoel) Mesostoma lingua the which blastomere (Costello and Henley 1976; Verdonk pharynx primordium is formed at mid-levels along the and van den Biggelaar 1983; Henry and Martindale 1998; antero-posterior axis (Bresslau 1904; Hartenstein and Boyer et al. 1996, 1998; Henry et al. 2000). Experimental Ehlers 2000), and in triclads (e.g., Schmidtea polychroa; manipulations and molecular studies have also revealed Bennazzi and Gremigni 1982) it defines the posterior pole. intrinsic cell fate determinants that are expressed at an The position of the pharynx primordium at early stages early stage in a distinct blastomere and are then dis- thus closely corresponds to the position where the pharynx tributed by means of an invariant pattern of cleavage di- ends up later. Aside from its location, the later small size visions to the different cells whose fate they control and low complexity of the Macrostomum pharynx is also (Bagu and Boyer 1990). As pointed out above, in reflected in its early appearance. This supports the notion Macrostomum sp., cleavage initially follows the conven- that the pharynx simplex coronatus, as defined by Doe tional pattern, but then departs from this pattern when (1981) for all Macrostomorpha, may be homologous to the certain blastomeres, most likely vegetal cells, stretch out other pharyngeal differentiations seen in higher rhabdi- around the remaining blastomeres to form the outer yolk tophoran flatworms (see Ehlers 1985; Rieger et al. 1991, mantle. It is currently not clear exactly which cells con- for summaries). Precursors of the massive arrays of radial tribute to the yolk mantle. Inverse epiboly begins at a muscle fibers characteristic of the pharynx bulbosus, the stage when only two to three micromere quartets are most advanced pharynx type, are visible from the very formed. Seilern-Aspang (1957) suggested that either all beginning; by contrast, in Macrostomum sp., pharynx four macromeres, or part of the macromeres plus part of muscle precursors in the pharynx simplex form only a the third quartet micromeres, give rise to the outer yolk thin, inconspicuous layer around the pharynx epithelium. mantle. In either way, the remaining blastomeres that be- The primordium of the body wall musculature in come surrounded by the external yolk need to generate all Macrostomum sp. first becomes apparent during stage 5 of the cell types, including the mesoderm and the gut, as an irregular, two to three cell diameters thick cell layer with a reduced number of macromeres or no macromeres underneath the epidermis. A series of block-shaped my- at all. Once surrounded by the yolk mantle, cell divisions oblasts have been reported for M. hystricinum marinum in these inner blastomeres (the embryonic primordium) (Reiter et al. 1996). These authors observed a close as- are difficult to observe. So far, we have no observations sociation between myoblasts and neuroblasts during dif- that any sort of spiral pattern is resumed; mitotic spindles ferentiation, perhaps reflecting the intricate functional appear to be random, and cell sizes initially do not suggest relationship of muscle and nerve cells in the body-wall a distinction between macromeres and micromeres. Dye musculature. Myoblasts subsequently flatten, elongate injection-based lineage studies are required to follow later and send out muscle processes. Nothing can be said cleavage and to reconstruct lineage relationships. It will currently about the earlier origin of muscle precursors. In also be interesting to establish how the expression of larval polyclads there is a different origin of longitudinal specific determinants in Macrostomum sp. has adapted to and circular muscles, the longitudinal fibers deriving from the novel types of lineages. For example, are endo- and the vegetal 4d blastomere called ento-mesoderm, the mesodermal fate determinants, which in conventional circular fibers from cells deriving from the 2b micromere, spiralians appear in the macromeres and in specific fourth called ecto-mesoderm or ecto-mesenchyme (Boyer et al. quartet micromeres, not expressed in early vegetal blas- 1996). In Acoela all muscle cells are apparently derived tomeres, but rather come on at a later stage in distinct from the ento-mesoderm (Henry et al. 2000). Dye injec- locations within the embryonic primordium? We expect tion-based lineage tracing experiments will be required to that molecular-genetic studies will shed light on this and verify whether a similar distinction also exists for Mac- related questions that are important in reconstructing the rostomum sp. Furthermore, we have recently cloned the evolution of developmental mechanisms. homolog of the muscle specification gene Mef 2 (Morris et al., in preparation) which, in both vertebrates and Dro- sophila, is expressed in mesoderm, followed by muscle Organogenesis from organ primordia cells. Macrostomum sp. Mef 2 is expressed specifically in the adult musculature, and it is hoped that in situ hy- The developmental steps that lead from the early embry- bridization to early embryos will provide us with a more onic primordium of Macrostomum sp. to the body of the detailed picture of the origin of muscles in this species. hatching juvenile correspond closely in most important Specific molecular markers will also be needed to aspects to what has been described for other flatworm make progress in understanding the origin of the central taxa. Thus, following the establishment of the undiffer- nervous system in Macrostomum sp. or any other flat- entiated embryonic primordium, individual organ pri- worm. Neuronal precursors can only be distinguished mordia appear. The first organ primordia invariably are from myoblasts at the time when cell differentiation sets those of the brain and the pharynx; in Macrostomum sp., in, and neurons send out axons (see Reiter et al. 1996; as in the dalyellid (rhabdocoel) Gieysztoria superba and Rieger 1998). The tyrTub marker allows one to study late the temnocephalid (rhabdocoel) Craspedella pedum,the aspects of neural differentiation (similar to acTub in other 238 species; Younossi-Hartenstein et al. 2000; Younossi- Bagu J, Carranza S, Paps J, Ruiz-Trillo I, Riutort M (2001) Hartenstein and Hartenstein 2001), but it does not help to Molecular and phylogeny of the Tricladida. 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