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 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 animals. 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 planarians, 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 Macrostomorpha. 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