The exceptional stem cell system of Macrostomum lignano: Screening for gene expression and studying cell proliferation by hydroxyurea treatment and irradiation

Daniela Pfister¹ , Katrien De Mulder¹^,2 , Isabelle Philipp¹ , Georg Kuales¹ , Martina Hrouda¹ , Paul Eichberger³ , Gaetan Borgonie² , Volker Hartenstein⁴ and Peter Ladurner¹

¹Institute of Zoology, University of Innsbruck. Technikerstrasse 25, A-6020 Innsbruck, Austria

²Department of Biology, University of Ghent, Ledeganckstraat 35, B-9000 Ghent, Belgium

³Department of Radiotherapy and Radiation Oncology, University Hospital Innsbruck, Medical University Innsbruck. Anichstrasse 35, A-6020 Innsbruck, Austria

⁴Department of Molecular, Cell and Developmental Biology, University of California Los Angeles, Los Angeles, CA 90095, USA

author email corresponding author email

Frontiers in Zoology 2007, 4:9doi:10.1186/1742-9994-4-9


Published:	9 March 2007

Abstract

Background

Flatworms are characterized by an outstanding stem cell system. These stem cells (neoblasts) can give rise to all cell types including germ cells and power the exceptional regenerative capacity of many flatworm species. Macrostomum lignano is an emerging model system to study stem cell biology of flatworms. It is complementary to the well-studied planarians because of its small size, transparency, simple culture maintenance, the basal taxonomic position and its less derived embryogenesis that is more closely related to spiralians. The development of cell-, tissue- and organ specific markers is necessary to further characterize the differentiation potential of flatworm stem cells. Large scale in situ hybridization is a suitable tool to identify possible markers. Distinguished genes identified in a large scale screen in combination with manipulation of neoblasts by hydroxyurea or irradiation will advance our understanding of differentiation and regulation of the flatworm stem cell system.

Results

We have set up a protocol for high throughput large scale whole mount in situ hybridization for the flatworm Macrostomum lignano. In the pilot screen, a number of cell-, tissue- or organ specific expression patterns were identified. We have selected two stem cell- and germ cell related genes – macvasa and macpiwi – and studied effects of hydroxyurea (HU) treatment or irradiation on gene expression. In addition, we have followed cell proliferation using a mitosis marker and bromodeoxyuridine labeling of S-phase cells after various periods of HU exposure or different irradiation levels. HU mediated depletion of cell proliferation and HU induced reduction of gene expression was used to generate a cDNA library by suppressive subtractive hybridization. 147 differentially expressed genes were sequenced and assigned to different categories.

Conclusion

We show that Macrostomum lignano is a suitable organism to perform high throughput large scale whole mount in situ hybridization. Genes identified in such screens – together with BrdU/H3 labeling – can be used to obtain information on flatworm neoblasts.