Cell and Tissue Research

, 327:637 | Cite as

Regeneration in Macrostomum lignano (Platyhelminthes): cellular dynamics in the neoblast stem cell system

  • Katharina Theresia Nimeth
  • Bernhard Egger
  • Reinhard Rieger
  • Willi Salvenmoser
  • Roland Peter
  • Robert Gschwentner
Regular Article


Neoblasts are potentially totipotent stem cells and the only proliferating cells in adult Platyhelminthes. We have examined the cellular dynamics of neoblasts during the posterior regeneration of Macrostomum lignano. Double-labeling of neoblasts with bromodeoxyuridine and the anti-phospho histone H3 mitosis marker has revealed a complex cellular response in the first 48 h after amputation; this response is different from that known to occur during regeneration in triclad platyhelminths and in starvation/feeding experiments in M. lignano. Mitotic activity is reduced during the first 8 h of regeneration but, at 48 h after amputation, reaches almost twice the value of control animals. The total number of S-phase cells significantly increases after 1 day of regeneration. A subpopulation of fast-cycling neoblasts surprisingly shows the same dynamics during regeneration as those in control animals. Wound healing and regeneration are accompanied by the formation of a distinct blastema. These results present new insights, at the cellular level, into the early regeneration of rhabditophoran Platyhelminthes.


Regeneration S-phase Cell cycle Planarian Macrostomum lignano (Platyhelminthes) 



We are grateful to Gunde Rieger for critical comments on earlier drafts of the manuscript and to Peter Ladurner for stimulating discussions. We thank Professor B. Pelster, Institute of Zoology and Limnology, Innsbruck, for allowing us to use his confocal microscope.


  1. Agata K (2003) Regeneration and gene regulation in planarians. Curr Opin Genet Dev 13:492–496PubMedCrossRefGoogle Scholar
  2. Agata K, Watanabe K (1999) Molecular and cellular aspects of planarian regeneration. Semin Cell Dev Biol 10:377–383PubMedCrossRefGoogle Scholar
  3. Baguñà J (1974) Dramatic mitotic response in planarians after feeding, and a hypothesis for the control mechanism. J Exp Zool 190:117–122PubMedCrossRefGoogle Scholar
  4. Baguñà J (1976) Mitosis in the intact and regenerating planarian Dugesia mediterranea n. sp. II. Mitotic studies during regeneration, and a possible mechanism of blastema formation. J Exp Biol 195:65–80Google Scholar
  5. Baguñà J (1981) Planarian neoblasts. Nature 290:14–15CrossRefGoogle Scholar
  6. Baguñà J (1998) Planarians. In: Ferretti P, Geraudie J (eds) Cellular and molecular basis of regeneration: from invertebrates to human. Wiley, Chichester, pp 135–166Google Scholar
  7. Baguñà J, Riutort M (2004a) Molecular phylogeny of the Platyhelminthes. Can J Zool 82:168–193CrossRefGoogle Scholar
  8. Baguñà J, Riutort M (2004b) The dawn of bilaterian animals: the case of acoelomorph flatworms. BioEssays 26:1046–1057PubMedCrossRefGoogle Scholar
  9. Baguñà J, Romero R (1981) Quantitative analysis of cell types during growth, degrowth and regeneration in the planarians Dugesia mediterranea and Dugesia tigrina. Hydrobiologia 84:181–194CrossRefGoogle Scholar
  10. Baguñà J, Saló E, Auladell C (1989) Regeneration and pattern formation in planarians. III. Evidence that neoblasts are totipotent stem cells and the source of blastema cells. Development 107:77–86Google Scholar
  11. Baguñà J, Romero R, Saló E, Collet J, Auladell C, Ribas M, Riutort M, Garcia-Fernandez J, Burgaya F, Bueno D (1990) Growth, degrowth and regeneration as developmental phenomena in adult freshwater planarians. In: Marthy HJ (ed) Experimental embryology in aquatic plants and animals. Plenum, New York, pp 129–162Google Scholar
  12. Baguñà J, Saló E, Romero R, Garcia-Fernandez J, Bueno D, Muñoz-Marmol AM, Bayascas-Ramirez JR, Casali A (1994) Regeneration and pattern formation in planarians: cells, molecules and genes. Zool Sci 11:781–795Google Scholar
  13. Best JB, Hand S, Rosenvold R (1968) Mitosis in normal and regenerating planarians. J Exp Biol 168:157–168Google Scholar
  14. Bode A, Salvenmoser W, Nimeth K, Mahlknecht M, Adamski Z, Rieger RM, Ladurner P (2006) The neoblast stem cells of Macrostomum sp. (Platyhelminthes): ultrastructure of immunogold labeled S-phase cells and non S-phase neoblasts, their numbers and distribution. Cell Tissue Res (in press)Google Scholar
  15. Brøndsted H (1969) Planarian regeneration. Pergamon, Oxford LondonGoogle Scholar
  16. Carranza S, Baguñà J, Riutort M (1997) Are the Platyhelminthes a monophyletic primitive group? An assessment using 18S rDNA sequences. Mol Biol Evol 14:485–497PubMedGoogle Scholar
  17. Cook CE, Jimenez E, Akam M, Saló E (2004) The Hox gene complement of acoel flatworms, a basal bilaterian clade. Evol Dev 6:154–163PubMedCrossRefGoogle Scholar
  18. Drobysheva IM (1986) Physiological regeneration of the digestive parenchyma in Convoluta pulchra and Oxyposthia praedator (Turbellaria, Acoela). Cytometry 132:189–193Google Scholar
  19. Dubois F (1949) Contribution à l’étude de la migration des cellules de régénération chez les planaires dulcicoles. Bull Biol Fr Belg 83:213–283Google Scholar
  20. Egger B, Ladurner P, Nimeth K, Gschwentner R, Rieger R (2006a) The regeneration capacity of the flatworm Macrostomum lignano - on repeated regeneration, rejuvenation, and the minimal size needed for regeneration. Dev Genes Evol, DOI 10.1007/s00427-006-0069-4
  21. Egger B, Ladurner P, Nimeth K, Gschwentner R, Rieger R (2006b) The regeneration capacity of the flatworm Macrostomum lignano - on repeated regeneration, rejuvenation, and the minimal size needed for regeneration. Dev Genes Evol, DOI 10.1007/s00427-006-0079-2 (erratum)
  22. Ehlers U (1985) Das Phylogenetische System der Plathelminthes. Fischer, StuttgartGoogle Scholar
  23. Gabriel A (1970) Etude morphologique et évolution biochimique des néoblastes au cours des premières phases de la régénération des planaires d´eau douce. Annls Embryol Morphogen 3:49–69Google Scholar
  24. Gremigni V, Miceli C (1980) Cytophotometric evidence for cell “transdifferentiation” in planarian regeneration. Arch Entwm 188:107–113CrossRefGoogle Scholar
  25. Gschwentner R, Ladurner P, Nimeth K, Rieger R (2001) Stem cells in a basal bilaterian. S-phase and mitotic cells in Convolutriloba longifissura (Acoela, Platyhelminthes). Cell Tissue Res 304:401–408PubMedCrossRefGoogle Scholar
  26. Heidenhain M (1885)Eine neue Verwendung des Hämatoxylin. Archiv Mikrosk Anat 24:468–470CrossRefGoogle Scholar
  27. Hori I (1997) Cytological approach to morphogenesis in the planarian blastema. II. The effect of neuropeptides. J Submicrosc Cytol Pathol 29:91–97PubMedGoogle Scholar
  28. Hori I, Kishida Y (1998) A fine-structural study of regeneration after fission in the planarian Dugesia japonica. Hydrobiologia 383:131–136CrossRefGoogle Scholar
  29. Hori I, Hikosaka-Katayama T, Kishida Y (1999) Cytological approach to morphogenesis in the planarian blastema. III. Ultrastructure and regeneration of the acoel turbellarian Convoluta naikaiensis. J Submicrosc Cytol Pathol 31:247–258Google Scholar
  30. Ito H, Saito Y, Watanabe K, Orii H (2001) Epimorphic regeneration of the distal part of the planarian pharynx. Dev Genes Evol 211:2–9PubMedCrossRefGoogle Scholar
  31. Ladurner P, Rieger R (2000) Embryonic muscle development of Convoluta pulchra (Turbellaria-Acoelomorpha, Platyhelminthes). Dev Biol 222:359–375PubMedCrossRefGoogle Scholar
  32. Ladurner P, Rieger R, Baguñà J (2000) Spatial distribution and differentiation potential of stem cells in hatchlings and adults in the marine platyhelminth Macrostomum sp.: a bromodeoxyuridine analysis. Dev Biol 226:231–241PubMedCrossRefGoogle Scholar
  33. Lindh NO (1957) The mitotic activity during the early regeneration in Euplanaria polychroa. Arkiv Zool 10:497–509Google Scholar
  34. Mollenhauer HH (1964) Plastic embedding mixtures for use in electron microscopy. Stain Technol 39:111–114PubMedGoogle Scholar
  35. Montgomery JR, Coward SJ (1974) On the minimal size of a planarian capable of regeneration. Trans Am Microsc Soc 93:386–391PubMedCrossRefGoogle Scholar
  36. Moore J, Willmer P (1997) Convergent evolution in invertebrates. Biol Rev Cambridge Philos Soc 72:1–60CrossRefPubMedGoogle Scholar
  37. Newmark PA, Sánchez Alvarado A (2000) Bromodeoxyuridine specifically labels the regenerative stem cells of planarians. Dev Biol 220:142–153PubMedCrossRefGoogle Scholar
  38. Newmark PA, Sánchez Alvarado A (2002) Not your father’s planarian: a classic model enters the era of functional genomics. Nat Rev Genet 3:210–219PubMedCrossRefGoogle Scholar
  39. Nimeth K, Ladurner P, Gschwentner R, Salvenmoser W, Rieger R (2002) Cell renewal and apoptosis in Macrostomum sp. (Lignano). Cell Biol Int 26:801–815PubMedCrossRefGoogle Scholar
  40. Nimeth KT, Mahlknecht M, Mezzanato A, Peter R, Rieger R, Ladurner P (2004) Stem cell dynamics during growth, feeding and starvation in the basal flatworm Macrostomum sp. (Platyhelminthes). Dev Dyn 230:91–99PubMedCrossRefGoogle Scholar
  41. Palmberg I (1986) Cell migration and differentiation during wound healing and regeneration in Microstomum lineare (Turbellaria). Hydrobiologia 132:181–188CrossRefGoogle Scholar
  42. Palmberg I (1990) Stem cells in microturbellarians: an autoradiographic and immunocytochemical study. Protoplasma 158:109–120CrossRefGoogle Scholar
  43. Pasquinelli AE, McCoy A, Jimenez E, Saló E, Ruvkun G, Martindale MQ, Baguñà J (2003) Expression of the 22 nucleotide let-7 heterochronic RNA throughout the Metazoa: a role in life history evolution? Evol Dev 5:372–378PubMedCrossRefGoogle Scholar
  44. Peter R (2001) Experimentelle Systeme zum Studium von Regenerationsvorgängen: Turbellarien als Modellorganismen mit einem Stammzellensystem. Ber Nat-Med Verein Innsbruck 88:287–350Google Scholar
  45. Peter R, Ladurner P, Rieger RM (2001) The role of stem cell strategies in coping with environmental stress and choosing between alternative reproductive modes: Turbellaria rely on a single cell type to maintain individual life and propagate species. Mar Ecol PSZNI 22:35–51CrossRefGoogle Scholar
  46. Peter R, Gschwentner R, Schürmann W, Rieger RM, Ladurner P (2004) The significance of stem cells in free-living flatworms: one common source for all cells in the adult. J Appl Biomed 2:21–35Google Scholar
  47. Reddien PW, Oviedo NJ, Jennings JR, Jenkin JC, Sánchez Alvarado A (2005) SMEDWI-2 is a PIWI-like protein that regulates planarian stem cells. Science 310:1327–1330PubMedCrossRefGoogle Scholar
  48. Reuter M, Kreshchenko N (2004) Flatworm asexual multiplication implicates stem cells and regeneration. Can J Zool 82:334–356CrossRefGoogle Scholar
  49. Rieger R (1996) Plathelminthes, Plattwürmer. In: Westheide W, Rieger R (eds) Spezielle Zoologie. I. Einzeller und Wirbellose Tiere. Fischer, Stuttgart, pp 210–258Google Scholar
  50. Rieger RM, Gehlen M, Haszprunar G, Holmlund M, Legniti A, Salvenmoser W, Tyler S (1988) Laboratory cultures of marine Macrostomida (Turbellaria). Forts Zool 36:525Google Scholar
  51. Rieger RM, Salvenmoser W, Legniti A, Tyler S (1994) Phalloidin-rhodamine preparations of Macrostomum hystricinum marinum (Plathelminthes): morphology and postembryonic development of the musculature. Zoomorphology 114:133–147CrossRefGoogle Scholar
  52. Rieger RM, Legniti A, Ladurner P, Reiter D, Asch E, Salvenmoser W, Schürmann W, Peter R (1999) Ultrastructure of neoblasts in microturbellaria: significance for understanding stem cells in free-living Platyhelminthes. Invertebr Repr Dev 35:127–140Google Scholar
  53. Saló E, Baguñà J (1984) Regeneration and pattern formation in planarians. I. The pattern of mitosis in anterior and posterior regeneration in Dugesia (G) tigrina, and a new proposal for blastema formation. J Embryol Exp Morphol 83:63–80PubMedGoogle Scholar
  54. Saló E, Baguñà J (2002) Regeneration in planarians and other worms: new findings, new tools, and new perspectives. J Exp Zool 292:528–539PubMedCrossRefGoogle Scholar
  55. Salvenmoser W, Riedl D, Ladurner P, Rieger R (2001) Early steps in the regeneration of the musculature in Macrostomum sp. (Macrostomorpha, Platyhelminthes). Belg J Zool 131 (Suppl 1):105–109Google Scholar
  56. Salvetti A, Batistoni R, Deri P, Rossi L, Sommerville J (1998) Expression of DjY1, a protein containing a cold shock domain and RG repeat motifs, is targeted to sites of regeneration in planarians. Dev Biol 201:217–229PubMedCrossRefGoogle Scholar
  57. Salvetti A, Rossi L, Deri P, Batistoni R (2000) An MCM2-related gene is expressed in proliferating cells of intact and regenerating planarians. Dev Dyn 218:603–614PubMedCrossRefGoogle Scholar
  58. Sánchez Alvarado A, Newmark PA, Robb SMC, Juste R (2002) The Schmidtea mediterranea database as a molecular resource for studying platyhelminthes, stem cells and regeneration. Development 129:5659–5665PubMedCrossRefGoogle Scholar
  59. Telford MJ, Lockyer AE, Cartwright-Finch C, Littlewood DT (2003) Combined large and small subunit ribosomal RNA phylogenies support a basal position of the acoelomorph flatworms. Proc R Soc Lond [Biol] 270:1077–1083CrossRefGoogle Scholar
  60. Tyler S (1976) Comparative ultrastructure of adhesive systems in the Turbellaria. Zoomorphologie 84:1–76Google Scholar
  61. Tyler S (2001) The early worm: origins and relationship of the lower flatworms. In: Littlewood DTJ, Bray RA (eds) Interrelationships of the Platyhelminthes. Taylor & Francis, New York, pp 3–12Google Scholar

Copyright information

© Springer-Verlag 2006

Authors and Affiliations

  • Katharina Theresia Nimeth
    • 1
  • Bernhard Egger
    • 1
  • Reinhard Rieger
    • 1
  • Willi Salvenmoser
    • 1
  • Roland Peter
    • 2
  • Robert Gschwentner
    • 1
  1. 1.Institute of ZoologyUniversity of InnsbruckInnsbruckAustria
  2. 2.Department of Genetics and General BiologyUniversity of SalzburgSalzburgAustria

Personalised recommendations