Advancing the flatworm Macrostomum lignano as a versatile model organism for stem cell research

Flatworms are a classical model for regeneration and stem cell research due to their astonishing regeneration capacity facilitated by pluripotent stem cells called neoblasts. M. lignano has several important experimental properties that make it a convenient model organism: the transparency of the animal facilitates observation of morphological changes at different conditions and treatments, whereas short generation time, ease of culturing and high number of progeny, combined with available draft genome and transcriptome data and established protocols for RNA interference (RNAi), in situ hybridization (ISH) and BrdU labeling provide the basis for genomics approaches in this organism. Upon amputation, M. lignano is able to regenerate the whole posterior part, including gonads and other organs, from a small head stump, and thus it provides a powerful experimental system to study the molecular mechanisms governing regeneration and function of neoblasts. Although substantial progress has been made in understanding neoblast regulation, the absence of stable transgenic lines and genome editing tools impeded a detailed functional analysis and stem cell lineage tracing. The work described in this thesis is the result of efforts to explore the concept of adult pluripotent stem cells and to establish robust methods for genome engineering in the flatworm Macrostomum lignano. Chapter 1 gives a general introduction for the concept of the stem cells, their regulation and stem cells in flatworms. Chapter 2 describes establishment of transgenic lines in Macrostomum lignano by transposase approach. In Chapter 3 effects of Hippo pathway on homeostasis and regeneration of a highly plastic organism, Macrostomum lignano, is demonstrated. We show the regulatory roles of Hippo pathway in proliferation and selfrenewal of neoblasts. Chapter 4 provides a proof of principle for the first successful genome engineering in flatworms. Efficient site specific gene knock out and knock in via TALENs is achieved. In Chapter 5 regulatory roles of DDX39, ATP dependent RNA helicase, on proliferation is demonstrated in both flatworms and cell line. This study suggests conserved functions of DDX39 among species and convenience of using Macrostomum lignano as a complementary model to mammals. Finally in Chapter 6 a general discussion about the achieved goals during this PhD and possible future directions of the studies on Macrostomum lignano is discussed.