Cell and Tissue Research

, Volume 347, Issue 2, pp 397–406 | Cite as

Proliferative response of the stem cell system during regeneration of the rostrum in Macrostomum lignano (Platyhelminthes)

  • Freija Verdoodt
  • Wim Bert
  • Marjolein Couvreur
  • Katrien De Mulder
  • Maxime Willems
Regular Article
First Online:
Received:
Accepted:

Abstract

Macrostomum lignano (Platyhelminthes) possesses pluripotent stem cells, also called neoblasts, which power its extraordinary regeneration capacity. We have examined the cellular dynamics of neoblasts during regeneration of the rostrum in M. lignano. First, using live squeeze observations, the growth curve of the rostrum was determined. Second, neoblasts were labelled with 5-bromo-2'-deoxyuridine (BrdU) and an anti-phospho-histone H3 mitosis marker (anti-phos-H3) to analyze their proliferative response to amputation. During the regeneration process, both S- and M-phase cells were present anterior to the eyes, a region that is devoid of proliferating cells during homeostasis. Furthermore, BrdU pulse experiments revealed a biphasic S-phase pattern, different from the pattern known to occur during regeneration of the tail plate in M. lignano. During a first systemic phase, S-phase numbers significantly increased, both in the region adjacent to the wound (the anterior segment) and the region far from the wound (the posterior segment). During the second, spatially restricted phase, S-phase numbers in the anterior segment rose to a peak at 3 to 5 days post-amputation (p-a), while in the posterior segment, S-phase activity approached control values again. A blastema, characterized as a build-up of S- and M-phase cells, was formed 1 day p-a.

Altogether, our data present new insights into the cellular response of the neoblast system upon amputation, clearly demonstrating important differences from the situation known to occur during regeneration of the tail plate. Furthermore, the presence of proliferating cells in the region anterior to the eyes shows a clear alteration in stem cell regulation during regeneration.

Keywords

Macrostomum lignano Neoblasts Platyhelminthes S-phase Regeneration 
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Notes

Acknowledgements

We want to thank Bernhard Egger for advice concerning the BrdU labellings and for useful comments and discussions. Carl Vangestel is acknowledged for advice on statistical methods. Furthermore, the authors thank the anonymous reviewers for their insightful comments and suggestions, which have led to a significant improvement of the manuscript. FV and MW received funding from the Institute for the Promotion of Innovation through Science and Technology in Flanders (IWT-Vlaanderen).

Supplementary material

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Supplementary Fig. 1

Scanning electron microscopy of the wound after amputation of the rostrum in M. lignano. a,b Scanning electron microscopic images of the rostrum region in M. lignano. a Control animal. Lateral view on the rostrum area. Ciliae of epidermal cells (arrowheads) and longer ciliae of sensory nerves (open arrowheads) are visible. Anterior is on top, ventral side is on the left. b One hour p-a. View on the anterior tip of the regenerating rostrum. The wound area is almost completely covered with a layer of epidermal cells, leaving only a small opening (asterisk). Ventral is on the left/bottom. Bars 10 μm

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Supplementary Fig. 2

Immunostaining of muscle F-actin (phalloidin) after amputation of the rostrum in M. lignano. Confocal projections of the rostrum area after immunostaining of muscle F-actin, using TRITC-conjugated phalloidin. a Control animal, dorsal view. b One hour p-a, ventral view. Contraction of muscle fibers is visible at the level of the wound, resulting in a reduction of the wound area. Arrows indicate the level of the eyes. Anterior is on top. Bars 10 μm

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Supplementary Fig. 3

Spatial distribution of S-phase cells during regeneration of the rostrum in M. lignano (BrdU pulse). BrdU labelling after a 30-minute BrdU pulse. a-i Confocal projections of the rostrum area. a Two hours p-a. b Eight hours p-a. c Three days p-a. d Four days p-a. e Five days p-a. f Seven days p-a. g Eight days p-a. h Nine days p-a. i Ten days p-a. Arrows indicate the level of the eyes. Anterior is on top. Bars 50 μm

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Supplementary Fig. 4

Immunostaining of the serotonergic nervous system (anti 5-HT) after amputation of the rostrum in M. lignano. Confocal projections of the rostrum after immunostaining of neurons expressing the neuropeptide serotonin. a Control animal. Two condensed clusters of serotonergic neurons are present at the lateral sides of the neuropile (asterisks). Emanating from the neuropile are axons, which form a subepidermal plexus (see also Morris et al. 2007). b Zero hours p-a. The condensed clusters at the lateral sides of the neuropile are not damaged when amputation is performed at the indicated cutting level. c Two hours p-a. Neurons of the subepidermal plexus are present in the anterior tip of the rostrum. Arrows indicate the level of the eyes. Anterior is on top. Bars 10 μm

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Copyright information

© Springer-Verlag 2012

Authors and Affiliations

  • Freija Verdoodt
    • 1
  • Wim Bert
    • 1
  • Marjolein Couvreur
    • 1
  • Katrien De Mulder
    • 2
  • Maxime Willems
    • 3
  1. 1.Nematology Unit, Department of BiologyGhent UniversityGhentBelgium
  2. 2.Department of Clinical Chemistry, Microbiology, and ImmunologyGhent UniversityGhentBelgium
  3. 3.Department of Pharmaceutics, Laboratory of Pharmaceutical TechnologyGhent UniversityGhentBelgium

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