Invertebrate Reproduction and Development, 35 :2 (1999) 127-140 Balaban, Philadelphia/Rehovot 0168-8170/99/$05.00 Q 1999 Balaban I A! A! Ultrastructure of neoblasts in microturbellaria: significance for understanding stem cells in free-living Platyhelminthes REINHARD M. RIEGER'*, ALEXANDER LEGNITI', PETER LADURNER', DIETMAR REITER', ESTHER ASCH', / WILLIBALD SALVENMOSER', WOLFGANG SCH~RMANN~and ROLAND PETER^ t 'Institutfir Zoologie und Limnologie, Universitat Innsbruck, Technikersn: 25, A-6020 Innsbruck Austria Email: reinhard.rieger@uibkac. at 2~nstitutfirGenetik und Allgemeine Biologie, Universitdt Salzburg, Hellbrunnersh: 34, A-5020 Salzburg, Austria Received 18 December 1998; Accepted 16 January 1999 Summary Platyhelminths possess a unique stem cell system that is claimed to be totipotent. It is supposed to be competent for the renewal of all cell types, including germ cells, during postembryonic development and regeneration. A connection to stem cells in the embryo has been postulated repeatedly. This cell type is now most frequently termed "neoblast". Light microscopy can reveal only a few neoblast characters, and ultrastructural studies have shown additional characters for discriminating possible types and/or stages. While some progress has been made in this respect for triclad turbellarians (freshwater planarians), rather little is known about the microturbellarians. We have investigated the fine structure of neoblasts of hatchlings and adults of Macrostomum hystricinum marinum, a member of a primitive taxon in the "Turbel1aria"- Rhabditophora (a paraphyletic group giving rise to the main parasitic flatworm taxa). In t Macrostomum, one population of neoblasts is located in lateral bands along the main longitudinal nerve cords within the body cavity. Another population is found in the gastrodermis in a basi-epithelial position. Based on their cytoplasmic and nuclear organization, three stages 3 in neoblast differentiation have been distinguished. The first and second stages are characterized 14, by cytoplasm lacking organelles except free ribosomes and scattered mitochondria, a finding 1 identical with the picture known from the "classical" planarian neoblast. In the first stage, heterochromatin is scattered over the nucleus in isolated clumps in a typical speckled (checkerboard) appearance; a nuclear lamina is weakly developed. In stage 2 the hetero- chromatin forms strands and clumps connected to each other. In stage 3 the nucleus is characterized by more prominent heterochromatin strands and by heterochromatin attachments to the well developed nuclear lamina. In this last stage a rough endoplasmic reticulum (rER) and Golgi complex are also present, indicating the entrance into cytoplasmic differentiation. Early epidermal replacement cells are located baso-epithelially, which show a nuclear organization similar to stage 3 neoblasts. Observations of stem cells in regenerating specimens and on isolated neoblasts are reported briefly. The data show that from the three types of differentiating cells distinguished recently in regenerative blastemas of planarians, the first stage ("undifferentiated cells") resembles stage 2 neoblasts described here for postembryonic development. The results are compared with observations that have been published for neoblasts in other free-living platyhelminths. *correspondingauthor. 128 R.M. Rieger et al. / IRD 35 (1999) 127-140 Key woruk: Stem cells, neoblasts, ultrastructure, heterochromatin, Platyhelminthes Introduction BagufIB et a]., 1990, 1994; Bagufii, 1998). Data on The stem cells in Platyhelminthes, usually called neoblasts in microturbellarians, especially with respect neoblasts, are the only known source for cell additions to their ultrastructure, are rare and have been during growth and regeneration (Gustafsson, 1977; documented only in few cases (see Ehlers, 1985; Bagufii, 1981; Ehlers, 1985; Palmberg, 1991; BagufiB Rieger, 1985; Rieger eta]., 1991a; Ehlers, 1992). More et al., 1990, 1994; Hori, 1997; Baguiii, 1998). Such a detailed studies (Palmberg, 1990, 1991) have been single-stem cell type for all tissues represents a rather published only for Microstomum (Macrostomorpha). In extraordinary situation in the Animal Kingdom. In this paper we want to provide a first overview of the general, progenitor cells or stem cells (for terminology ultrastructural characteristics of the neoblast system in see Gardner and Beddington, 1988; Morrison et al., the macrostomomorphan genus Macrostomum because - 1997; Wolpert, 1998) are involved in these processes the Macrostomorpha represent one of the most [ primitive taxa of the Rhabditophora (Rieger et al, - - in organisms of many metazoan taxa (Potten, 1983; Lord and Dexter, 1988), but rarely are such cells the 199 1a, Ehlers, 1995). One major technical advantage only source of cell renewal during postembryonic life for choosing microturbellarianssuch as Macrostomum (Gilbert, 1997). or Microstomum for studying neoblasts is the possi- The earliest neoblasts are defined by a high nucleo- bility of labeling DNA by the incorporation of tritiated cytoplasmic ratio and a rim of extremely basophilic thymidine (Palmberg, 1991) or BrdU (Ladurner et al., cytoplasm rich in free ribosomes. The cells are 1998). These techniques have failed so far for spherical or ovoid. In spite of the great number of planarians (Bagufik pers. comm.). results in favour of their totipotency, little is known Taking into account the scarceness of light regarding the actual alterations of neoblasts during cell microscopical characters available for distinguishing cycles, e.g., of symmetrical or asymmetrical cell between subpopulations,nuclear structure seemed to us divisions. The existence of subtypes or different lines a promising candidate for fine tuning neoblast of such cells has been suspected and even postulated in characterization in addition to cytoplasmic structures models, especially since the neoblast pool of planarians (see also Morita et al., 1969; Hay and Coward, 1975). appears heterogeneous in light of cell kinetic data._ Heterochromatin has long been known to represent, to (BagufiA et al., 1990; BagufiA, 1998). Also separation a large proportion, either noncoding or genetically by density gradient centrifugation (Schiirmann et a]., inactive parts of the genome (see, e.g., Alberts et a]., 1988, 1995) has provided hints that subtypes of 1994). More recent findings have proven that inclusion , neoblasts might exist. It has been suggested repeatedly of genes in heterochromatic areas may well regulate - that neoblasts in the adult are derived from early gene expression (Elgin, 1996; Spector, 1996; Wolffe, embryonic stem cells because the cells resemble each 1998). A detailed analysis of the heterochromatin other (lit. in Rieger, 1985; Ehlers, 1985; BaguAa and pattern ofthe platyhelminths appears, therefore, helpful Boyer, 1990). The actual ontogenetic origin of for a still more detailed analysis of gene expression neoblasts is, however, still an open question. patterns among primitive Metazoa The universal role of the neoblast as a single-stem The Platyhelminthes feature among the most cell type is, in general, also ascribed to the generation primitive Bilateria as is expressed in most modern of germ cells (Baguila and Boyer, 1990). As a phylogenetic trees (Conway Morris, 1993; Ehlers, consequence, flatworms are generally supposed to lack 1995; Ax, 1996; Haszprunar, 1996; Carranza et a]., a separate germ line (for special cases see Gremigni, 1997; Ehlers and Ehlers, 1998). The central phylo- 1981, 1988). This is supported by the fact that the genetic position of platyhelminths makes the study of transition between sexual animals and asexual forms the neoblast cell system instrumental for generating completely lacking any gonads is common in ontogenetic baseline data in the Bilateria. In com- planarians (Ehlers, 1985; BaguAA et al., 1990, 1994). parison with cellular mechanisms known from stem This notion is still open for discussion. cell systems in adult and embryonic mammals (Potten The structure of neoblasts (including ultrastructure) and Morris, 1988; Heath and Smith, 1988), data on the is best known from studies of regeneration processes in much more primitive stem cell system of the Rhabditophora-Tricladida (planarians, see lit. in platyhelminths should give better insight into Pedersen, 1972; Hori, 1982; 1997; Ehlers, 1985; mechanisms involved. R.M. Rieger ef al. / IRD 35 (1 999) 127-1 40 129 Material and Methods Results Almost all observations are based on data from Observations on living specimens and cell Macrostomum hystricinum rnarinum Rieger 1977. se~arations Observations on living specimens and the studies on During light microscopical studies for the descrip- isolated neoblasts have been carried out with another, tion of the new Macrostomum species collected at yet undescribed species of the genus Macrostomum Lignano, Italy, spherical cells could be observed along collected in the northern Adriatic near Lignano, Italy. the lateral margins when interference contrast optics The average size of Mh. marinum is 0.7-1.4 mm and were applied to optimal squeeze preparations (Figs. 1, that ofMacrostomum sp. is 0.9-1.5 mm. Mh. marinum 2). The diameter of such cells ranged from 5 to 9 ym. came from field collections at the localities of the The fact that they had a very high nucleocytoplasmic original description (Rieger, 1977; Ladumer et al., ratio and a prominent nucleolus made them likely to be 1997) as well as from laboratory cultures. The fixation