Embryonic origins of hull cells in the flatworm Macrostomum lignano through cell lineage analysis: developmental and phylogenetic implications

  • Maxime WillemsEmail author
  • Bernhard Egger
  • Carsten Wolff
  • Stijn Mouton
  • Wouter Houthoofd
  • Pamela Fonderie
  • Marjolein Couvreur
  • Tom Artois
  • Gaëtan Borgonie
Original Article


The development of macrostomid flatworms is of interest for evolutionary developmental biology research because these taxa combine characteristics of the canonical spiral cleavage pattern with significant deviations from this pattern. One such deviation is the formation of hull cells, which surround the remaining embryonic primordium during early development. Using live observations with a 4D microscope system, histology, and 3D reconstructions, we analyzed the ontogeny of these hull cells in the macrostomid model organism Macrostomum lignano. Our cell lineage analysis allowed us to find the precursors of the hull cells in this species. We discuss the relation between macrostomid development and the development of other spiralians and the question of whether hull cells are homologous within rhabditophoran flatworms.


Embryo 4D microscopy Cell lineage Hull cells Ontogeny Rhabditophora 



M.W. would like to thank Prof. Dr. Dominique Adriaens for the use of the Amira software and Dr. David Weisblat for the help in improving the manuscript. This work was supported by IWT doctoral grants to M.W. and S.M. (by the Institute for the Promotion of Innovation through Science and Technology in Flanders (IWT-Vlaanderen)) and a FWO postdoctoral fellowship to W.H. B.E. was supported by a postdoctoral fellowship from the Francqui Fondation, and a Sparkling Science grant funded by the Austrian Ministry of Science and Research. C. W. would like to thank Volker Hartenstein for the opportunity to work on flatworms.

Supplementary material

Supplementary movie S1

Hull cell formation. Movie of the first 12 h of development. Blastomeres become less recognizable because hull cells start to surround them (AVI 8,879 kb)

Supplementary movie S2

Hull cell degeneration. The process starts approximately 51 h after egg laying. The movie covers 6 h of development. Arrows indicate areas of cellular immigration (AVI 0 kb)


  1. Ashburner M (1989) Drosophila. A laboratory manual. Cold Spring Harbor, Cold Spring Harbor, p 433Google Scholar
  2. Ax P (1961) Verwandtschaftsbeziehungen und Phylogenie der Turbellarien. Ergebn Biol 24:1–68PubMedGoogle Scholar
  3. Ax P, Borkott H (1968a) Organisation und Fortpflanzung von Macrostomum romanicum (Turbellaria, Macrostomida). Verh Dtsch Zool Ges Innsbruck 30b:344–347Google Scholar
  4. Ax P, Borkott H (1968b) Organisation und Fortpflanzung von Macrostomum salinum (Turbellaria-Macrostomida). Inst Wiss Film C 947:1–11Google Scholar
  5. Baguñà J, Boyer B (1990) Descriptive and experimental embryology of the Turbellaria: present knowledge, open questions and future trends. In: Marthy H (ed), Experimental embryology in aquatic plants and animals. NATO ASI Series A 195:95–128Google Scholar
  6. Baguñà J, Riutort M (2004) Molecular phylogeny of the Platyhelminthes. Can J Zool 82:168–193CrossRefGoogle Scholar
  7. Bebenek IG, Gates RD, Morris J, Hartenstein V, Jacobs DK (2004) Sine oculis in basal Metazoa. Dev Genes Evol 214:342–351CrossRefPubMedGoogle Scholar
  8. Bode A, Salvenmoser W, Nimeth K, Mahlknecht M, Adamski Z, Rieger RM, Peter R, Ladurner P (2006) Immunogold-labeled S-phase neoblasts, total neoblast number, their distribution, and evidence for arrested neoblasts in Macrostomum lignano (Platyhelminthes, Rhabditophora). Cell Tissue Res 325:577–587CrossRefPubMedGoogle Scholar
  9. Bogomolow S (1949) Zur Frage nach dem Typus der Furchung bei den Rhabdocoela. Wiss Schr Leningrader Staatl Univ Ser Biol 20:128–142Google Scholar
  10. Bogomolow S (1960) Über die Furchung von Macrostomum rossicum Beklemichev und deren Beziehung zur Furchung der Turbellaria, Coelata und Acoela. Vt Sov Can Embriol SSSR 1960:23–24Google Scholar
  11. Boyer BC, Henry JQ, Martindale MQ (1996) Dual origins of mesoderm in a basal spiralian: cell lineage analyses in the polyclad turbellarian Notoplana inquilina. Dev Biol 179:328–338CrossRefGoogle Scholar
  12. Boyer BC, Henry JQ, Martindale MQ (1998) The cell lineage of a polyclad turbellarian embryo reveals close similarity to coelomate spiralians. Dev Biol 204(1):111–123CrossRefPubMedGoogle Scholar
  13. Bresslau E (1904) Beitraege zur Entwicklungsgeschichte der Turbellarien. I. Die Entwicklung der Rhabdocoelen und Alloiocoelen. Z Wiss Zool 76:213–332Google Scholar
  14. Byrne M, Villinski JT, Cisternas P, Siegel RK, Popodi E, Raff RA (1999) Maternal factors and the evolution of developmental mode: evolution of oogenesis in Heliocidaris erythrogramma. Dev Genes Evol 209:275–283CrossRefPubMedGoogle Scholar
  15. Cardona A, Hartenstein V, Romero R (2005) The embryonic development of the triclad Schmidtea polychroa. Dev Genes Evol 215(3):109–131CrossRefPubMedGoogle Scholar
  16. Cardona A, Hartenstein V, Romero R (2006) Early embryogenesis of planaria: a cryptic larva feeding on maternal resources. Dev Genes Evol 216(11):667–681CrossRefPubMedGoogle Scholar
  17. Dohle W, Gerberding M, Hejnol A, Scholtz G (2004) Cell lineage, segment differentiation and gene expression in crustaceans. In: Scholtz G (eds), Evolutionary developmental biology of Crustacea A.A. Balkema Lisse, pp 95–133Google Scholar
  18. Dolinski C, Borgonie G, Schnabel R, Baldwin JG (1998) Buccal capsule development as a consideration for phylogenetic analysis of Rhabditida (Nemata). Dev Genes Evol 208:495–503CrossRefPubMedGoogle Scholar
  19. Dunn CW, Hejnol A, Matus DQ, Pang K, Browne WE, Smith SA, Seaver E, Rouse GW, Obst M, Edgecombe GD, Sørensen MV, Haddock SHD, Schmidt-Rhaesa A, Okusu A, Kristensen RM, Wheeler WC, Martindale MQ, Giribet G (2008) Broad taxon sampling improves resolution of the Animal Tree of Life in phylogenomic analyses. Nature 452:745–750CrossRefPubMedGoogle Scholar
  20. Eisenman EA, Alfert M (1982) A new fixation procedure for preserving the ultrastructure of marine invertebrate tissues. J Microsc 125:117–120Google Scholar
  21. Egger B, Ishida S (2005) Chromosome fission or duplication in Macrostomum lignano (Macrostomorpha, Plathelminthes) – Remarks on chromosome numbers in "archoophoran turbellarians". J Zool Syst Evol Res 43:127–132CrossRefGoogle Scholar
  22. Egger B, Ladurner P, Nimeth K, Gschwentner R, Rieger R (2006) The regeneration capacity of the flatworm Macrostomum lignano—on repeated regeneration, rejuvenation, and the minimal size needed for regeneration. Dev Genes Evol 216:565–580CrossRefPubMedGoogle Scholar
  23. Egger B, Steinke D, Tarui H, De Mulder K, Arendt D, Borgonie G, Funayama N, Gschwentner R, Hartenstein V, Hobmayer B, Hooge M, Hrouda M, Ishida S, Kobayashi C, Kuales G, Nishimura O, Pfister D, Rieger R, Salvenmoser W, Smith J Jr, Technau U, Tyler S, Agata K, Salzburger W, Ladurner P (2009) To be or not to be a flatworm: the acoel controversy. PLoS ONE 4(5):e5502CrossRefPubMedGoogle Scholar
  24. Giesa S (1966) Die Embryonalentwicklung von Monocelis fusca Oersted (Turbellaria, Proseriata). Z Morphol Oekol Tiere 57:137–230CrossRefGoogle Scholar
  25. Hejnol A, Schnabel R (2005) The eutardigrade Thulinia stephaniae has an indeterminate development and the potential to regulate early blastomere ablations. Development 132:1349–1361CrossRefPubMedGoogle Scholar
  26. Hejnol A, Schnabel R (2006) What a couple of dimensions can do for you: comparative developmental studies using 4D-microscopy- examples from tardigrade development. Integ Comp Biol 46:151–161CrossRefGoogle Scholar
  27. Hejnol A, Schnabel R, Scholtz G (2006) A 4D-microscopic analysis of the germ band in Porcellio scaber (Malacostraca, Peracarida)—developmental and phylogenetic implications. Dev Genes Evol 216:755–767CrossRefPubMedGoogle Scholar
  28. Henry JQ, Martindale MQ (1999) Conservation of the spiralian developmental program: cell lineage of the nemertean, Cerebratulus lacteus. Dev Biol 201:253–269CrossRefGoogle Scholar
  29. Henry JQ, Martindale MQ, Boyer BC (1995) Axial specification in a basal member of the spiralian clade: Lineage relationships of the first four cells to the larval body plan in the polyclad turbellarian Hoploplana inquilina. Biol Bull 189:194–195Google Scholar
  30. Houthoofd W, Jacobsen K, Mertens C, Vangestel S, Coomans A, Borgonie G (2003) Embryonic cell lineage of the marine nematode Pellioditis marina. Dev Biol 258:57–69CrossRefPubMedGoogle Scholar
  31. 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(2):231–241CrossRefPubMedGoogle Scholar
  32. Ladurner P, Schärer L, Salvenmoser W, Rieger RM (2005) A new model organism among the lower Bilateria and the use of digital microscopy in taxonomy of meiobenthic Platyhelminthes: Macrostomum lignano, n. sp. (Rhabditophora, Macrostomorpha). J Zool Sys Evol Res 43(2):114–126CrossRefGoogle Scholar
  33. Lambert JD (2008) Mesoderm in spiralians: the organizer and the 4d cell. J Exp Zoolog B Mol Dev Evol 308B:15–23CrossRefGoogle Scholar
  34. Morris J, Nallur R, Ladurner P, Egger B, Rieger R, Hartenstein V (2004) The embryonic development of the flatworm Macrostomum sp. Dev Genes Evol 214:220–239CrossRefPubMedGoogle Scholar
  35. Morris J, Ladurner P, Rieger R, Pfister D, Jacobs D, Hartenstein V (2006) The Macrostomum lignano EST database as a molecular resource for studying platyhelminth development and phylogeny. Dev Genes Evol 216:695–707CrossRefPubMedGoogle Scholar
  36. Nimeth K, Ladurner P, Gschwenter R, Salvenmoser W, Rieger R (2002) Cell renewal and apoptosis in Macrostomum lignano [Lignano]. Cell Biol Int 26(9):801–815CrossRefPubMedGoogle Scholar
  37. Nimeth K, Mahlknecht M, Mezzanato A, Peter R, Rieger R, Ladurner P (2004) Stem cell dynamics during growth, feeding and starvation in the basal flatworm Macrostomum lignano (Platyhelminthes). Dev Dyn 230:91–99CrossRefPubMedGoogle Scholar
  38. Papi F (1953) Beitraege zur Kenntnis der Macrostomiden (Turbellarien). Acta Zool Fenn 78:1–32Google Scholar
  39. Peter R, Ladurner P, Rieger R (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 22:35–45CrossRefGoogle Scholar
  40. Peter R, Gschwentner R, Schurmann W, Rieger R, Ladurner P (2004) The significance of stem cells in free-living flatworms: one common source for all cells in the adult. J Appl Med 2:21–35Google Scholar
  41. Pfister D, De Mulder K, Philipp I, Kuales G, Hrouda M, Eichberger P, Borgonie G, Hartenstein V, Ladurner P (2007) The exceptional stem cell system of Macrostomum lignano: Screening for gene expression and studying cell proliferation by hydroxyurea treatment and irradiation. Frontiers in Zoology 4:9CrossRefPubMedGoogle Scholar
  42. Philippe H, Brinkmann H, Martinez P, Riutort M, Baguñà J (2007) Acoel flatworms are not platyhelminthes: evidence from phylogenomics. PLoS ONE 2(1):717CrossRefGoogle Scholar
  43. Reisinger E (1923) Turbellaria. In: Schulze (ed), Biologie der Tiere Deutschlands. pp 1–64Google Scholar
  44. Rieger RM, Gehlen M, Haszprunar G, Holmlund M, Legniti A, Salvenmoser W, Tyler S (1988) Laboratory cultures of marine Macrostomida (Turbellaria). Fortschr Zool 36:523Google Scholar
  45. Robert A (1903) Recherches sur le developpement des Troques. Arch Zool Exp 3e Se´r 10:269–538Google Scholar
  46. Salvenmoser W, Riedl D, Ladurner P, Rieger R (2001) Early steps in the regeneration of the musculature in Macrostomum lignano (Macrostomorpha, Platyhelminthes). Belg J Zool 131:63–67Google Scholar
  47. Schärer L, Ladurner P (2003) Phenotypically plastic adjustment of sex allocation in a simultaneous hermaphrodite. ProcRoy Soc London B 270:935–941CrossRefGoogle Scholar
  48. Schärer L, Joss G, Sandner P (2004a) Mating behaviour of the marine turbellarian Macrostomum sp.: these worms suck. Marine Biology 145:373–380CrossRefGoogle Scholar
  49. Schärer L, Ladurner P, Rieger R (2004b) Bigger testes do work more: experimental evidence that testis size reflects testicular cell proliferation activity in the marine invertebrate, the freeliving flatworm Macrostomum sp. Behav Ecol Sociobiol 56:420–425CrossRefGoogle Scholar
  50. Schärer L, Sandner P, Michiels N (2005) Trade-off between male and female allocation in the simultaneously hermaphroditic flatworm Macrostomum sp. J Evol Biol 18:396–404CrossRefPubMedGoogle Scholar
  51. Schnabel R, Hutter H, Moerman D, Schnabel H (1997) Assessing normal embryogenesis in Caenorhabditis elegans using a 4D microscope: variability of development and regional specification. Dev Biol 184:234–265CrossRefPubMedGoogle Scholar
  52. Schockaert E, Hooge M, Sluys R, Schilling S, Tyler S, Artois T (2008) Global diversity of free living flatworms (Platyhelminthes,"Turbellaria") in freshwater. Hydrobiologia 595:41–48CrossRefGoogle Scholar
  53. Scholtz G (2005) Homology and ontogeny: pattern and process in comparative developmental biology. Theory Biosci 124:121–143PubMedGoogle Scholar
  54. Seilern-Aspang F (1957) Die Entwicklung von Macrostomum appendiculatum (Fabricius). Zool Jahrb Anat 76:311–330Google Scholar
  55. Sempere LF, Martinez P, Cole C, Baguñà J, Peterson KJ (2007) Phylogenetic distribution of microRNAs supports the basal position of acoel flatworms and the polyphyly of Platyhelminthes. Evol Dev 9(5):409–415PubMedGoogle Scholar
  56. Sulston JE, Horvitz HR (1977) Post-embryonic cell lineages of the nematode Caenorhabditis elegans. Dev Biol 82:41–55CrossRefGoogle Scholar
  57. Spurr AR (1969) A low-viscosity epoxy resin embedding medium for electron microscopy. J Ultrastruct Res 26:31–43CrossRefPubMedGoogle Scholar
  58. Surface FM (1907) The early development of a polyclad, Planocera inquilina. Wh Proc Acad Nat Sci Phila 59:514–559Google Scholar
  59. Thomas MB (1986) Embryology of the Turbellaria and its phylogenetic significance. Hydrobiologia 132:105–115CrossRefGoogle Scholar
  60. Tyler S (1981) Development of cilia in embryos of the turbellarian Macrostomum. Hydrobiologia 84:231–239CrossRefGoogle Scholar
  61. Urbach R, Schnabel R, Technau GM (2003) The pattern of neuroblast formation, mitotic domains and proneural gene expression during early brain development in Drosophila. Development 130:3589–3606CrossRefPubMedGoogle Scholar
  62. Wallberg A, Curini-Galletti M, Ahmadzadeh A, Jondelius U (2007) Dismissal of Acoelomorpha: Acoela and Nemertodermatida are separate early bilaterian clades. Zool Scr 36:509–523CrossRefGoogle Scholar
  63. Willems WR, Wallberg A, Jondelius U, Littlewood DTJ, Backeljau T, Schockaert ER, Artois T (2006) Filling a gap in the phylogeny of flatworms: relationships within the Rhabdocoela (Platyhelminthes), inferred from 18S ribosomal DNA sequences. Zool Scr 35(1):1–17CrossRefGoogle Scholar
  64. Wolff C, Scholtz G (2006) Cell lineage analysis of the mandibular segment of the amphipod Orchestia cavimana reveals that the crustacean paragnaths are sternal outgrowths and no limbs. Frontiers in Zoology 3(1):19CrossRefPubMedGoogle Scholar
  65. Wray GA, Bely AE (1994) The evolution of echinoderm development is driven by several distinct factors. Development 120:97–106Google Scholar
  66. Wray GA, Raff RA (1990) Novel origins of lineage founder cells in the direct developing sea urchin Heliocidaris erythrogramma. Dev Biol 141:41–54CrossRefPubMedGoogle Scholar
  67. Younossi-Hartenstein A, Hartenstein V (2000) The embryonic development of the polyclad flatworm Imogine mcgrathi. Dev Genes Evol 210:383–398CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag 2009

Authors and Affiliations

  • Maxime Willems
    • 1
    Email author
  • Bernhard Egger
    • 2
  • Carsten Wolff
    • 3
  • Stijn Mouton
    • 1
  • Wouter Houthoofd
    • 1
  • Pamela Fonderie
    • 1
  • Marjolein Couvreur
    • 1
  • Tom Artois
    • 4
  • Gaëtan Borgonie
    • 1
  1. 1.Department of BiologyGhent UniversityGhentBelgium
  2. 2.Institute of ZoologyUniversity of InnsbruckInnsbruckAustria
  3. 3.Institut für BiologieHumboldt-Universität zu Berlin,Vergleichende ZoologieBerlinGermany
  4. 4.Center for Environmental Sciences, Research Group Biodiversity Phylogeny and Population StudiesHasselt UniversityAgoralaanBelgium

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