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"- (a paraphyletic group giving rise to the main parasitic 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 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, 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 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 neoblasts. Similar spherical cells with the same range procedures used for specimens from natural of diameter and the same cytological features were populations have been described in Rieger and Ruppert isolated after disintegration of whole animals from the (1978), and those for the cultured animals in Rieger et same species (Fig. 3). The specific staining by azure A- al. (1991~). eosin B supports their neoblast nature. Most data were derived from serial transverse sections of tissue slices (2-10 ym thick) in the area at the end of the gut of hatchlings (see also Rieger et al., UItrastructure of neoblasts 1994). One juvenile animal (about 400pm long) The fine structure of neoblasts in Macrostomum collected in the field and four freshly hatched resembled that of other platyhelminth taxa in many specimens (about 300pm long) from the laboratory respects and especially showed many similarities to population were used for this purpose. All other cell planarian stem cells. A thorough analysis of nuclear types were recorded in these sections as well in order structures allowed a classification into three types that to guarantee a complete survey of all neoblasts. In have been interpreted as stages, based on the addition, we studied longitudinal and transverse single correlation with a progressive increase in cytoplasmic sections from various body regions of juvenile and organelles. They are described in more detail below, adult specimens. All sections were cut with diamond together with some peculiarities depending on their knives with an Ultracut E or Ultracut UCT and viewed location. So far, we have not observed structures that with a Zeiss 902 TEM. could be interpreted without doubt as chromatin For observations on living specimens, animals were satellite material or chromatoid bodies. This applies to anesthesized with MgCI, isotonic to seawater and "mesodermal" and gastrodermal neoblasts as well as to viewed through a Reichert Polyvar compound cells ofthe regeneration blastema and sharply contrasts microscope equipped with Nomarski interference with the situation in planarians. contrast optics. Isolated neoblasts were prepared by a modified method originally devised for planarian stem Neoblasts within "mesodermal" tissues cells (SchUrmann and peter, 1995, 1988). The method In this bbmesodermaltissue,, (for see allows staining for neoblasts with azure A-eosin Discussion section), neoblasts were concentrated in because of their basOphilic lateral bands along the main longitudinal nerve cords (Pedersen, 1959). in the body cavity. We observed a few mitotic Preliminar'Y experiments have been carried out on divisions; one such division in a neoblast adjacent to Macrostomum sp. were cut the protonephridial canal system is depicted in Fig. 8. with razor blades in mid-bOd~.Only the part From all characters taken together, the following three regenerated completely (see also ~ad~nleret al., 1997)- stages of neoblast differentiation could be dis- Twenty-four hours after cutting, anterior halves were tinguished. fixed in 2.5% glutaraldehyde buffered with 0.1 M Na- cacodylate containing 10% sucrose. Following post- Stage I: Neoblasts from freshly hatched specimens fixation in 1% osmium tetroxide in the same buffer and are usually oval, about 2pm wide and 4ym long standard dehydration in acetone, the specimens were (Fig. 4). In some juveniles where the volume of embedded in Spurr's low viscosity resin for the mesodermal tissue is relatively larger, spherical preparation of ultrathin sections to be viewed with the neoblasts occur (Fig. 5). The cytoplasm of these cells TEM. varied in size, but has always been found to contain R. M. Rieger et al. / 1RD 35 (1999) 127-1 40

Fig. 1. Macrostomum sp. (cultured specimen from Lignano, northern Adriatic). Anterior half of living animal, IC-contrast, viewed from ventral; note brain (br), diatom (di) in gut (g), eye (e), mouth opening (m). Inset between figures: whole animal, total length 1 mm; position of sections shown in Fig. 4 and 5 (arrowheads). Fig. 2. Macrostomum sp., enlargement of epidermis (ep), tissues in body cavity and gut (g) in living animal shown in Fig. 1; note round cells surmised as neoblasts, situated just below the epidermis (arrows), longitudinal muscle fiber of body wall (Imb), insunk rhabdite gland cell opening through lateral epidermis (rhb), rhammite gland cell opening at anterior tip of animal (rhm). .W

only free ribosomes and mitochondria. These charac- clearly different pattern: it is arranged in irregular ters that have been described for planarian neoblasts strands or irregular clumps, most of which are classify them as typical stem cells without signs of apparently connected with each other (Fig. 8; see also cytoplasmic differentiation. Fig. 14). The nuclear lamina is inconspicuous when The nuclear lamina in these cells is only faintly compared to that of nuclei of differentiated cells (e.g., visible and might be missing over large areas. Most of muscle cells) in the mesodermal tissue. notably, these early neoblasts exhibit a distinct organization of their heterochromatin: it is, for the Stage 3. This stage represent neoblasts already most part, restricted to isolated, small (0.2pm wide) committed to differentiation. In addition to ribosomes clumps that apparently are not connected to each other: and mitochondria, Golgi complex and rER were visible only a few, if any heterochromatin, are attached to the in the cytoplasm. .nuclear lamina. The nuclei show a speckled (checker- In the nuclei, a continuous layer of heterochromatin board) appearance when compared with differentiated could be found beneath the continuous nuclear cells (Figs. 6,7). envelope, in addition to irregular strands of hetero- chromatin crossing the nucleoplasm. These strands Fig Stage 2. In other neoblasts with the same appeared as irregular clumps in individual sections sac cytoplasmic organization, the heterochromatin shows a (Fig. 1 1). The heterochromatin pattern resembled that tiss R.M. Rieger et al. /IRD 35 (1999) 127-140 131

found in blastema cells of regenerating animals; in both types of neoblasts, differentiated ER and Golgi complex were present. Centrioles were seen lying adjacent to the nucleus in some cells.

Neoblasts within the gastrodermis Neoblasts in the gastrodermis were always located at the base of the epithelium. They were usually spherical and some cells showed basically the same fine structure as described above for stage 1 neoblasts in mesodermal tissue: they were 34pm in diameter and contained no cell organelles except ribosomes and mitochondria (Figs. 9, 10). Their nucleus showed the same structure as in stage 1 neoblasts\ located in mesodermal tissue; isolated clumps of heterochromatin were distributed in a regular pattern. Cells with rER were placed with stage 3; a nuclear lamina was clearly visible. The heterochromatin organization appeared, however, somewhat different from that in stage 3 neoblasts from mesodermal tissue in that the heterochromatin clumps were at least twice as large.

Epidermal replacement cells Fig. 3. Neoblasts (arrowheads) in cell suspension of disintegrated specimen of Macrostomum sp (cultured Only one cell in the epidermis could be identified as specimen from Livorno, northern Adriatic). an epidermal replacement cell at the beginning of

Fig 5.4. TEM cross section through hatchling of Macrostomum hystricinum marinum (from cultures) near caudal end of the sac:-like, ciliated gut (g); position of area indicated in Fig. 1; note location of neoblast stage 1 (arrowhead) in mesodermal tis!cue, profiles of main longitudinal nerve cord (mln) and protonephridia (small arrows). 132 R.M. Rieger et al. / IRD 35 (1999) 127-140

Fig. 5. TEM cross section through juvenile specimen of Macrostomum hystricinum marimim (field population) in the caudal- most region of the sac-like, ciliated gut (g); note progressive stages in the incorporation of new epidermal cells (arrows), cmb =circular muscle fiber ofthe body wall (cmb), circular muscle fiber of the gut (cmg), epidermis (ep), epidermal replacement cell (erc), longitudial muscle fiber of the body wall (lmb), main longitudinal nerve cord (mln), stage 1 neoblasts (nb 1). differentiation. This cell had a small cytoplasmic Within the blastema, we did not find any cells process reaching into underlying mesodermal tissue resembling stage 1 neoblasts described above. The (Fig. 12). A cluster of two to four centrioles in the majority of the blastema cells were spherical or oval cytoplasm adjacent to the overlying epidermal cells (4-7pm long and 2-3 pm wide), and contained - in was found in one of the sections through this cell. addition to free ribosomes and mitochondria - the This spherical cell was very different from the rER and Golgi complex (Fig. 13). Some cells tenta- mature epidermal cells with their finger-like basal tively classified as stage 2 neoblasts had only very little interdigitations (Fig. 6). The cross section of the ER and a nucleus with a thin nuclear lamina (Fig. 13). nucleus was round and thus clearly different from A number of degenerated cells (e.g., muscle cells) nuclei in differentiated epidermal cells. The latter exhibited an extremely dense heterochromatin pattern nuclei are conspicuous in their star-shaped or antler- in their nuclei. shaped forms and a distinct layer of heterochromatin beneath the nuclear lamina (Fig. 5). Germ cells Fig. 6. In adult specimens, the anterior-most region of the with r Blastemal cells in regenerating animals gonads contained young spermatogonia and oogonia gastro The anterior half of specimens, fixed 24 h after (Figs. 14 spg, 15 og). With respect to the organization (~1,te having been cut in the mid-body region, had developed of the cytoplasm and the heterochromatin pattern of the a small blastema under the epidermis that had closed nucleus, these germ cells resembled either stage 1 over the caudal wound. Early stages of immigration of neoblasts (the oogonia; Figs. 8,14) or stage 2 neoblasts epidermal replacement cells could be observed at that (the spermatogonia; Figs. 7, 15). The similarity in time. heterochromatin structure of spermatogonia and of a = JC U! 1 'a= 13 av. .(JA)lapool Ierluah '(qln) sal!pqeqmqn '(w)qaM leu!uual '(u) uo!: qapool Xre!l!o pgso~'(8wo)in8 aqljo raqy apsnur mln3~~'(9~3) Lpoq aqljo 1aqg alosnw mln31!3 '(8urq) s!uuapo~lm8 e!uc jo xpxu [eseq '(awq) s!uuap!da jo x!qeur lemq 'I! ahoqe !apnu leuuap!da pm! gal IE (um) snapnu [la3 apsnur yl!~ ayl qn~smalonu u! saouaJag!p amduroa .anss!l puuaposaur u! lselqoau 1 a8gs ayl8u!~oqs'9 '8!d woy juawa8mlu~-9 '%!A

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Fig. 7. Enlargement from Fig. 5; note three stage 1 neoblasts in mesodermal tissue (nb I), nuclear structure of one possible stage 3 neoblast (x), two differentiated muscle cells (arrow), gut (g). stage 2 neoblast adjacent to the gonad is evident in Fig. 14. It is noteworthy that, in spite of the general similarity to neoblast nuclear morphology, the organi- zation of the heterochromatin allowed a clear distinction of early male and female germ cells.

Fig. 8. Detail of lateral margin of cross section through hatchling of Macrostomum hystricinum marinum (cultures) Discussion with dividing neoblast in center and one stage 2 neoblast Location of neoblasts (nb 2) above; nuclei of differentiated muscle (mn), epidermal nuclei (en), main longitudinal nerve cord (mln), cross section As described earlier in a TEM study of muscle through protonephridial system (pr), centrioles (ce), differentiation (Rieger et al., 1994), two populations of chromosomes (ch). neoblasts may be distinguished in Macrostomum: one within the gastrodermis and one within the nephridial system (always located slightly dorsal to the "mesodermal tissue". The "mesodermal tissue" of main lateral nerve cord), the typical sac-gonads, and platyhelminths has been claimed to be of entomeso- the male .and female genital system are also located dermal and ectomesenchymal origin (Hyman, 1951 ; within this "mesodermal tissue". Salvini-Plawen and Splechtna, 1979; Rieger, 1985; Boyer et al., 1996, in press). It contains myocytes of the body wall (circular, diagonal and longitudinal Ultrastructural characterization of early neoblasts: fibers), myocytes of the gut (longitudinal and circular the heterochromatin pattern fibers), insunken gland cell somas, a few "fixed" In this investigation of Macrostomum, we observed parenchymal cells with mostly fluid-filled cytoplasm the checkerboard heterochromatin pattern in neoblasts Fig. (terminology see Rieger, 1985) and neoblasts (Figs. 4, with minimal differentiation of cytoplasm (stage 1 the 5; see also Legniti et al., 1989). The paired proto- neoblasts) in postembryonic tissues. However, we mar R. M. Rieger et al. / IRD 35 (1 999) 12 7-1 40 135

Fig. 9. Stage 1 neoblast (nbl) in the gastrodemis in cross section through hatchling of Macrostomum hystricinum marinum (field population). Fig. 10. Enlargement fiom Fig. 9; circular muscle fiber of the body wall (cmb), gut (g), longitudinal muscle fiber of the body wall (Imb), longitudinal muscle fiber of the gut (Img).

Fig. 12. Epidermal replacement cell of Macrostomum hystricinum marinum (cultures), enlargement from Fig. 5; note small process reaching below the level of the epidermis (arrow).

could not find such neoblasts in regeneration blastemas. This corresponds with the picture of the "undifferentiated cells" in the regeneration blastemas of planarians recently publishedby Hori (1997): all Fig. 11. Stage 3 neoblast (nb3) in mesodermal tissues near nuclei in the electron micrographs lack the checker- the pharynx of a hatchling Macrostomum hystricinum board pattern of the heterochromatin. The marinum (cultures). rbg rhabdite gland. "undifferentiated cells" are comparable to stage 2 136 R. M.Rieger et al. / IRD 35 (1 999) 12 7-1 40

is more difficult to decide based on present ultra- structural evidence (see Palmberg, 1990, 1991). From the descriptions and because of a TEM illustration of a possible neoblast corresponding to stage 1 in our terminology (Palmberg, 1991, Fig. 13), we do expect the same stage 1 neoblasts in Microstomum. We agree with Palmberg (1991) on the notion that such an early stage may represent the original pool of neoblasts providing the "new proliferating cells" in the animal. In this context, it is of interest to mention the very similar cytoplasmic and heterochromatin structure found for neoblasts in planarian tumors (Hall et al., 1986). A significant difference of Macrostomum stage 1 , neoblasts compared to other turbellarian neoblasts C should be recalled. Chromatoid bodies, the con- spicuous electron-dense material well known from nuclei in planarian neoblasts (see Hay and Coward, 1975; Hori, 1982; Morita et al., 1984; Auladell et al., 1993; Hori, 1997), could not be clearly identified in these animals. In microturbellarians, such chromatoid bodies have been described for Microstomum lineare (Palmberg, 1991).

Gastrodermal neoblasts From the freshwater microturbellarians Micro- stomum Iineare and Stenostomum leucops, Reuter and Palmberg (1987) have reported gastrodermal neoblasts ranging between the pictures of stages 2 (hetero- chromatin pattern) and 3 (presence of a Golgi complex) of this study. In Macrostomum we found, in addition, typical stage 1 neoblasts in the gastrodermis.@l This can be taken as an indication for the presence of a Fig. 13. Longitudinal section through caudal blastema separate neoblast population in the gut. It has to be between gut and epidermis. neoblast stage 3 (nb 3) and clarified in the future (e.g., by labelling cells with possible neoblast stage 2 (x) in the regenerative blastema, BrdU) whether these stage 1 neoblasts immigrate from degenerated muscle cells (mcp). the "mesodermal" tissue, a process that seems to occur in planarians (pers. comm., J. Baguiii). As an neoblasts of our study. On the other hand, the alternative, gastrodermal neoblasts might migrate conspicuous heterochromatin pattern has previously during early development from the gastrodermis into been described for the earliest neoblast stage by Morita "mesodermal" tissue as is the case in certain cnidarians et al. (1969) from tissue adjacent to planarian (e.g., see Thomas and Edwards, 1991). regeneration blastemas. A similar early stage with this pattern of heterochromatin, called "beta cell", has been seen in cells in regular planarian tissue (Hay and Structural similarities of neoblasts and germ cell Coward, 1975). Bagufia et al. (1990) pointed out that progenitors these "beta cells" are simply early neoblasts. From all The similarities found in nuclear structure in oogonia these data we deduce at this point that "undifferen- and spermatogonia compared to neoblasts support the tiated cells" in regeneration blastemas are comparable view of a common cellular basis for asexual and sexual to stage 2 neoblasts in postembryonic development. reproduction in lower metazoans. As has been Whether an identical stage 1 neoblast as described proposed recently (Rieger and Weyrer, 1998), three here for Macrostomum is present also in Microstomum rather than two cell types are involved in the R.M. Rieger et al. / IRD 35 (1999) 127-1 40 137

Figs. 14, 15. Frontal sections through male (left side) and female (right side) gonads in Macrostomum hystricinum marinum; in the male gonad spermatogonia (*), synaptonemal complexes (arrows), spermatozoa (sp) and stage 2 neoblast outside the gonad in mesodermal tissue (nb 2); observe early oogonium (*) in the female gonad. propagation of primitive metazoans: an asexual "spore" Microstomum (nuclear morphology was not described in addition to male and female gametes. The in detail) differing in either possessing a variable gemmulae, common in freshwater sponges and the number of centrioles or lacking these organelles. The neoblast system of platyhelminths, are just two absence of centrioles from stage 1 neoblasts in examples corroborating this view. Asexual repro- Macrostomum seems odd taking into account their duction in both taxa is based on some sort of stem cell supposed function as replicating stem cells. One might system that propagates asexually and may give rise to speculate that they form a primary stock of mitotically germ cells as well. quiescent stem cells replenishing the proliferative cell department only upon need. Also a mere stage in a permanently self-renewing pool of stem cells would be Neo)blast ultrastructure and cell kinetics compatible with our observations. At present, we think IIn a study of neoblasts in Microstomum, Palmberg that our "neoblast stage 1 " represents the starting point ,. - (1 Y' 90, 1991) was the first to find evidence that of stem cell differentiation (see models of neoblast ultr astructural characters might be correlated with cell differentiation in BaguAa et al., 1990). However, even kinetics. She specified two types of neoblasts in information on symmetry or asymmetry of divisions is 138 R. M Rieger et al. / IRD 35 (1 999) 127-1 40 missing (see Gilbert, 1997; Morrison et al., 1997 for only a limited extent. This justifies an intensified general literature). application of other techniques, including genetic Tracing cell fates by labelling could provide a markers (Bagufib et al., 1994) and conventional and promising tool for following the pathways of neoblast immunocytochemical ultrastructural methods for a differentiation. Tritiated thymidine has been incor- more detailed understanding of platyhelminth develop- porated in Microstomum lineare in regenerating ment and regeneration in molecular terms. animals (Palmberg, 1990, 1991). The recent discovery that 5'-bromo-2-deoxyuridine (BrdU) labels Acknowledgements proliferating cells in Macrostomum (Ladumer et al., 1998) opens a further way of tracing cell fates with We acknowledge help during the preparation of the high precision. In addition, we now have successfully manuscript and its illustrations by Marijke de Jong- used antibodies against histones for labeling mitotic Brink, Gunde Rieger, Jaume BaguiiA, Robert stages (unpublished results by P. Ladumer, Gschwentner and Doris Riedl. M. Mahlknecht and A. Mezzanato). A quantitation of the heterochromatin fraction based on ultrastructural results could provide a still more precise indicator of References the differentiation stage. 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