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


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.

This is a preview of subscription content, log in to check access.

Access options

Buy single article

Instant unlimited access to the full article PDF.

US$ 39.95

Price includes VAT for USA

Subscribe to journal

Immediate online access to all issues from 2019. Subscription will auto renew annually.

US$ 99

This is the net price. Taxes to be calculated in checkout.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6


  1. Ashburner M (1989) Drosophila. A laboratory manual. Cold Spring Harbor, Cold Spring Harbor, p 433

  2. Ax P (1961) Verwandtschaftsbeziehungen und Phylogenie der Turbellarien. Ergebn Biol 24:1–68

  3. Ax P, Borkott H (1968a) Organisation und Fortpflanzung von Macrostomum romanicum (Turbellaria, Macrostomida). Verh Dtsch Zool Ges Innsbruck 30b:344–347

  4. Ax P, Borkott H (1968b) Organisation und Fortpflanzung von Macrostomum salinum (Turbellaria-Macrostomida). Inst Wiss Film C 947:1–11

  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 195:95–128

  6. Baguñà J, Riutort M (2004) Molecular phylogeny of the Platyhelminthes. Can J Zool 82:168–193

  7. Bebenek IG, Gates RD, Morris J, Hartenstein V, Jacobs DK (2004) Sine oculis in basal Metazoa. Dev Genes Evol 214:342–351

  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 325:577–587

  9. Bogomolow S (1949) Zur Frage nach dem Typus der Furchung bei den Rhabdocoela. Wiss Schr Leningrader Staatl Univ Ser Biol 20:128–142

  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 1960:23–24

  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 179:328–338

  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):111–123

  13. Bresslau E (1904) Beitraege zur Entwicklungsgeschichte der Turbellarien. I. Die Entwicklung der Rhabdocoelen und Alloiocoelen. Z Wiss Zool 76:213–332

  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 209:275–283

  15. Cardona A, Hartenstein V, Romero R (2005) The embryonic development of the triclad Schmidtea polychroa. Dev Genes Evol 215(3):109–131

  16. Cardona A, Hartenstein V, Romero R (2006) Early embryogenesis of planaria: a cryptic larva feeding on maternal resources. Dev Genes Evol 216(11):667–681

  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 95–133

  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 208:495–503

  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 452:745–750

  20. Eisenman EA, Alfert M (1982) A new fixation procedure for preserving the ultrastructure of marine invertebrate tissues. J Microsc 125:117–120

  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 43:127–132

  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 216:565–580

  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):e5502

  24. Giesa S (1966) Die Embryonalentwicklung von Monocelis fusca Oersted (Turbellaria, Proseriata). Z Morphol Oekol Tiere 57:137–230

  25. Hejnol A, Schnabel R (2005) The eutardigrade Thulinia stephaniae has an indeterminate development and the potential to regulate early blastomere ablations. Development 132:1349–1361

  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 46:151–161

  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 216:755–767

  28. Henry JQ, Martindale MQ (1999) Conservation of the spiralian developmental program: cell lineage of the nemertean, Cerebratulus lacteus. Dev Biol 201:253–269

  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 189:194–195

  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 258:57–69

  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):231–241

  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):114–126

  33. Lambert JD (2008) Mesoderm in spiralians: the organizer and the 4d cell. J Exp Zoolog B Mol Dev Evol 308B:15–23

  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 214:220–239

  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 216:695–707

  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):801–815

  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 230:91–99

  38. Papi F (1953) Beitraege zur Kenntnis der Macrostomiden (Turbellarien). Acta Zool Fenn 78:1–32

  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 22:35–45

  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 2:21–35

  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 4:9

  42. Philippe H, Brinkmann H, Martinez P, Riutort M, Baguñà J (2007) Acoel flatworms are not platyhelminthes: evidence from phylogenomics. PLoS ONE 2(1):717

  43. Reisinger E (1923) Turbellaria. In: Schulze (ed), Biologie der Tiere Deutschlands. pp 1–64

  44. Rieger RM, Gehlen M, Haszprunar G, Holmlund M, Legniti A, Salvenmoser W, Tyler S (1988) Laboratory cultures of marine Macrostomida (Turbellaria). Fortschr Zool 36:523

  45. Robert A (1903) Recherches sur le developpement des Troques. Arch Zool Exp 3e Se´r 10:269–538

  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 131:63–67

  47. Schärer L, Ladurner P (2003) Phenotypically plastic adjustment of sex allocation in a simultaneous hermaphrodite. ProcRoy Soc London B 270:935–941

  48. Schärer L, Joss G, Sandner P (2004a) Mating behaviour of the marine turbellarian Macrostomum sp.: these worms suck. Marine Biology 145:373–380

  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 56:420–425

  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 18:396–404

  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 184:234–265

  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 595:41–48

  53. Scholtz G (2005) Homology and ontogeny: pattern and process in comparative developmental biology. Theory Biosci 124:121–143

  54. Seilern-Aspang F (1957) Die Entwicklung von Macrostomum appendiculatum (Fabricius). Zool Jahrb Anat 76:311–330

  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):409–415

  56. Sulston JE, Horvitz HR (1977) Post-embryonic cell lineages of the nematode Caenorhabditis elegans. Dev Biol 82:41–55

  57. Spurr AR (1969) A low-viscosity epoxy resin embedding medium for electron microscopy. J Ultrastruct Res 26:31–43

  58. Surface FM (1907) The early development of a polyclad, Planocera inquilina. Wh Proc Acad Nat Sci Phila 59:514–559

  59. Thomas MB (1986) Embryology of the Turbellaria and its phylogenetic significance. Hydrobiologia 132:105–115

  60. Tyler S (1981) Development of cilia in embryos of the turbellarian Macrostomum. Hydrobiologia 84:231–239

  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 130:3589–3606

  62. Wallberg A, Curini-Galletti M, Ahmadzadeh A, Jondelius U (2007) Dismissal of Acoelomorpha: Acoela and Nemertodermatida are separate early bilaterian clades. Zool Scr 36:509–523

  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):1–17

  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):19

  65. Wray GA, Bely AE (1994) The evolution of echinoderm development is driven by several distinct factors. Development 120:97–106

  66. Wray GA, Raff RA (1990) Novel origins of lineage founder cells in the direct developing sea urchin Heliocidaris erythrogramma. Dev Biol 141:41–54

  67. Younossi-Hartenstein A, Hartenstein V (2000) The embryonic development of the polyclad flatworm Imogine mcgrathi. Dev Genes Evol 210:383–398

Download references


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.

Author information

Correspondence to Maxime Willems.

Additional information

Maxime Willems, Bernhard Egger, and Carsten Wolff equally contributed to this work.

Communicated by D.A. Weisblat

Electronic supplementary material

Below is the link to the electronic supplementary material.

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)

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)

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)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Willems, M., Egger, B., Wolff, C. et al. Embryonic origins of hull cells in the flatworm Macrostomum lignano through cell lineage analysis: developmental and phylogenetic implications. Dev Genes Evol 219, 409 (2009) doi:10.1007/s00427-009-0304-x

Download citation


  • Embryo
  • 4D microscopy
  • Cell lineage
  • Hull cells
  • Ontogeny
  • Rhabditophora