University of Groningen

The flatworm puzzleGrudniewska, Magda

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Citation for published version (APA):Grudniewska, M. (2017). The flatworm puzzle: Uncovering the molecular basis of the remarkable resilienceand regenerative capacity of Macrostomum lignano. University of Groningen.

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CHAPTER 1

General introduction and outline of the thesis

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1Model organisms in research

‘For a large number of problems, there will be some animal of choice, or a few such animals on which it can most conveniently be studied’

August Krogh (1929)

During the last decade, an impressive, rapid progress in Next Generation Se-quencing (NGS) technologies has been made. Initiated by the great success of the first assembly of the human genome project in 2003, we are witnessing an advent of these techniques. The combination of reduced costs and increased robustness, quality and sensitivity allows us to better address and understand the biology of complex organ-isms (Goodwin et al., 2016). Although NGS offers an enormous amount of generated data, in the majority of cases, experimental studies on model systems are still essential to understand how particular alterations at the DNA, RNA, or protein level affect func-tions of cells, tissues and finally, the entire organism. Such a model can be an animal, plant or microbe that can be used to study certain biological processes. In other words: it is a simplified system that is accessible and easily manipulated (Leonelli and Ankeny, 2013; De Magalhães, 2015). It is important, however, that data generated by studying a particular model system is applicable to other, often more complex organisms, such as Homo sapiens. Ethical concerns, high costs and technical limitations significantly reduce the experimental potential of humans, therefore, employing animal models as a proxy to study human biology is an essential part of modern research (Hunter, 2008).

Albeit the use of model organisms dates back to ancient times, it is the pioneer-ing work of George Mendel, in which he employed model systems such as peas or mice, to address fundamental questions about genetic heredity, that is considered as the first modern demonstration of the immense power of model organisms in biology (Müller and Grossniklaus, 2010). Since then, the preponderance of our knowledge about genet-ics, development, anatomy, physiology and various molecular and cellular processes is, indeed, obtained from studies of reference organisms.

The choice of the model organism is often determined by a particular question that researchers are striving to answer. In general, a good biological system should en-compass several attributes: (1) ease of breeding and maintaining in large numbers in

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the laboratory setting (i.e. short generation time and high fertility rates), (2) occupancy of a vital position in the phylogenetic tree, with a considerable degree of similarity to the organism we wish to reference the results to (i.e. identifiable human homologues genes), (3) having a well-established genetic background, and, consequently, (4) amena-bility to genetic manipulation (Govind, 2011; Hedges, 2002; Leonelli and Ankeny, 2013; Müller and Grossniklaus, 2010).

The ‘Big Five’ of traditional model organisms

There are several well-established and traditionally used animal models, such as yeast (Saccharomyces cerevisiae), the nematode worm (Caenorhabditis elegans), the fruit fly (Drosophila melanogaster), the zebrafish (Danio rerio) and the mouse (Mus mus-culus) (Minelli and Baedke, 2014). Each of these species possesses a unique set of at-tributes, creating an attractive platform to study a wide range of biological phenomena including development and stem cell functioning, ageing, regeneration, and cancer, to name only a few (Müller and Grossniklaus, 2010).

Indeed, several critical discoveries in fundamental biology are attributable to each of these models. A few seminal findings derived from studies in yeast include ad-vances in understanding the cell cycle, unraveling the function of telomeres and telo-merase and describing mechanism of autophagy (Guo-Lian et al., 1990; Hartwell et al., 1970; Lee and Nurse, 1987; Szostak and Blackburn, 1982; Takeshige et al., 1992).

Research using C. elegans contributed enormously to the progress in the field of genetic regulation, programmed cell death, discovery of RNA interference, or intro-ducing a green fluorescent protein as a marker for gene expression (Chalfie et al., 1994; Ellis and Horvitz, 1986; Fire et al., 1998).

It was over 100 years ago, when Thomas H. Morgan chose D. melanogaster to address the basic principles of genetic inheritance. Since then, discoveries in fruit flies have greatly advanced our understanding of the genetic control of early embryonic de-velopment, mechanisms of DNA damage or deciphering secrets of immunity (Lemaitre et al., 1996; Morgan, 1910; Muller, 1927; Nüsslein-Volhard and Wieschaus, 1980).

Another animal model, zebrafish, proved to be very powerful to study fun-damental aspects of vertebrate development (Keller et al., 2008). It also facilitates re-search in regenerative medicine and serves as a model for a number of human disorders (Lieschke and Currie, 2007; Poss et al., 2002).

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1Finally, the mouse is undisputedly the leading mammalian and prime model

to study the biology of stem cells. Furthermore, mice proved to be very robust in can-cer research or the studies of mechanistic underpinnings of metabolic, developmental, neurological and immune disorders (Cheng et al., 2010; Fuchs, 2005; Phifer-Rixey and Nachman, 2015; Snippert and Clevers, 2011).

A new era of research: nontraditional models in the spotlight

As illustrated above, traditional model organisms were and still are crucial for breakthroughs as well as steady advances in biology. They became the essential tools for biomedical research. Paradoxically, in line with the famous quote of Aristotle’s ‘The more you know, the more you know you don’t know’, the wealth of knowledge that is attributable to the use of the established model systems, created a number of new que-ries. Ironically, the standard models are often not the most appropriate reference to address questions from these emerging research areas (Austad, 2009, 2010; Goldstein and King, 2016). Fortunately, another characteristic of modern research is the rapid progress of new, high-throughput technologies, such as NGS, proteomics or targeted ge-nome editing (e.g. CRISP/Cas9), which are often applicable to diverse organisms. Con-sequently, scientists are no longer restrained to a handful of traditional model systems with well-established toolboxes (Goodwin et al., 2016; Hsu et al., 2014).

Nowadays, considerable attention is given to animals defined as experimental organisms or nontraditional models. They are often chosen to investigate a specific phe-nomenon, which they seem very suitable for. After all, the use of a study platform should be indeed dictated by specificity and uniqueness of the organism, and its characteristics relevant to studying a particular process or disease (Leonelli and Ankeny, 2013). Maybe they do not have all the attributes of model organisms, nor might they ever become one, but this should certainly not discourage researchers. The great value of these experi-mental systems is already widely recognized (Goldstein and King, 2016). For instance, the Nobel Prize laureates, Liz Blackburn and Joe Gall, emphasize the importance of reaching out to nontraditional models: ‘Biology sometimes reveals its general principles through that which appears to be arcane and even bizarre’ (Blackburn et al., 2006).

Therefore, to address the limitations of the traditional animal models on emerging research questions, and to concentrate on the high demand for alternative perspectives, the range of organisms used for biomedical research has greatly expand-ed and, most probably, will continue to grow (Figure 1). Several of such nontraditional systems are described below.

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Figure 1. Graphical representation of the most commonly used nontraditional ex-perimental organisms.

Tardigrades

A great example of an emerging, nontraditional reference animal is the tardi-grade (phylum: Tardigrada) (Fig. 1A). While initially considered as a good platform for space research, recently these little animals capture the attention of scientists because of their utility for biomedicine (Erdmann and Kaczmarek, 2016; Jönsson, 2007). Re-markably, the invertebrate phylum Tardigrada tolerates exposure to complete desic-cation, severe cold, and extreme galactic radiation. These impressive abilities inspired researchers to explore the therapeutic potential of key proteins responsible for the as-tonishing resistance of tardigrades to extreme conditions. A recent study by Hashimoto and colleagues provides a very promising perspective on the use of these animals to enhance the radiotolerance of human cells (Hashimoto et al., 2016). Although limited to few studies, the evidence that tardigrades may become a powerful experimental model in biomedicine is convincing (Goldstein and King, 2016; De Magalhães, 2015).

extremotolerant

short-living

immortal

astonishing regeneration ability

Macrostomum lignanoSchmidtea mediterranea

Nothobranchius furzeri Hydra vulgaris

Ramazzottius varieornatus

ageing extremesA B

C

Ambystoma mexicanum

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1Killifish

The killifish (Nothobranchius furzeri, phylum: Chordata) (Fig. 1B), a rising star in vertebrate ageing research, offers an alternative, very powerful platform to e.g. un-derstand how genes affect longevity or how regeneration capacity changes with age (Harel et al., 2015). The fish has an exceptionally short lifespan (3-7 months), most like-ly determined by environmental constraints of the natural habitat of the animal, and in consequence, it is the shortest-living vertebrate in the laboratory setting (Valdesalici and Cellerino, 2003). Since its introduction into biomedical research, it has been well characterized: identification of various ‘hallmarks of ageing’, indications that ageing of N. furzeri resembles that of mammals to a large extent, a high quality genome assem-bly and available genomic engineering tools, makes the killifish a powerful animal for systematic studies on ageing in a more convenient manner than in any other vertebrate model system (Harel et al., 2015; Hartmann and Englert, 2012; Platzer and Englert, 2016; Reichwald et al., 2015; Valenzano et al., 2011, 2015).

Axolotls

Among the vertebrates, the salamander species axolotl (Ambystoma mexica-num, phylum: Chordata) is unmatched in its regeneration capacity (Fig. 1C). The animal can regenerate multiple structures such as limbs, tail, jaw, ocular tissues, the intestine, small sections of the heart, spinal cord and parts of the brain (Amamoto et al., 2016; Brockes, 2005; Hosh et al., 1994). Methods developed specifically for axolotls, such as the live tracking system of fluorescently labeled cells (e.g. single muscle fibers), facil-itate studies of the precise mechanisms guiding regrowth of missing organs. For in-stance, in the seminal study of Kragl et al., it was shown that during limb regeneration, adult tissue is converted into a blastema, a region of undifferentiated progenitors with restricted potential, that gives a rise to the diverse tissues of the limb during regener-ation (Kragl et al., 2009). Elegant methods such as this example, show the great value of the axolotl to decipher the mechanistic underpinnings of regenerating complicated anatomical structures. (Sugimoto et al., 2011; Tanaka and Reddien, 2011).

Hydra

Another nontraditional model, with a great potential in ageing and regenera-tion studies, is Hydra (phylum: Cnidaria) (Fig. 1B), for which compelling evidence of a non-senescent life trajectory exists (Martínez, 1998; Schaible et al., 2015). This aston-ishing escape from senescence is believed to be attributable to the population of three distinct, multipotent stem cell types: the ectodermal and endodermal epithelial stem

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cells, and the interstitial stem cell lineage, that altogether can give rise to both somatic and germline cells (Bosch and David, 1987; Hobmayer et al., 2012). On the mechanistic level, it is postulated that the impressive regenerative abilities of the organism, constant and rapid cell turnover, may play a central role in its ‘immortality’ (Dańko et al., 2015; Martinez and Bridge, 2012; Schaible et al., 2015). This unique set of characteristics, combined with simple anatomy and the availability of an attractive toolbox, creates ex-citing opportunity to investigate the most enigmatic phenomena in biomedicine: regen-eration and immortality (Galliot, 2012; Gierer, 2012; Jones et al., 2014; Tomczyk et al., 2015).

Planarians

Alike Hydra, flatworms (phylum: Platyhelminthes) are recognizable models in stem cell, regeneration and ageing studies (Aboobaker, 2011; Rink, 2013; Sánchez Al-varado, 2006; Tanaka and Reddien, 2011) (Fig. 1C). Introduced over a century ago by Randolph and Morgan (Morgan, 1898; Randolph, 1897), research using flatworms has particularly expanded during the last two decades. Albeit several hundred species are known, the planarians (order: Tricladida) Dugesia japonica and Schmidtea mediterra-nea are the most widely studied animals. These free-living organisms have a bilater-al symmetry and relatively complex anatomy with distinct tissues and internal organs (e.g. nervous system, eyes, musculature, epidermis, gut) (Reddien and Sánchez Alvara-do, 2004). They are simple to maintain in the laboratory setting; the ability to reproduce in an asexual manner and a rapid development allow fast expansion of the experimental culture. The genome of S. mediterranea and the transcriptome of a few planarian spe-cies became available (Robb et al., 2008, 2015; Sánchez Alvarado et al., 2002; Shibata et al., 2012).

What makes flatworms particularly interesting, is their astonishing regener-ation competence (Rink, 2013). An adult individual, cut into as many as 279 pieces, were able to regrow an entire organism from each of these fragments (Morgan, 1901). This profound ability is facilitated by the population of actively dividing cells, called neoblasts, located in the mesodermal layer and distributed throughout the planarian body. Neoblasts constitute 20-30% of the cells in the adult animal and are described as the only somatic proliferative cells present in the organism. They give rise to the prog-eny that replace damaged or aged cells and replenish missing tissue (Aboobaker, 2011; Baguñà, 2012; Baguna and Romero, 1981; González-Estévez et al., 2012; Oviedo et al., 2003; Rink, 2013; Wagner et al., 2011). A landmark study by Baguna et al. (1989) in-volved the transplantation of neoblasts into irradiated, and thus stem cell devoid sexual and asexual strains of S. mediterranea. Interestingly, such a transplant of nonirradiated

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1tissue rescued the lethal phenotype and restored the regeneration capacity of the irra-diated animal. In other words, the neoblast population of S. mediterranea has a plurip-otent character (Baguñà et al., 1989). Another, more recent, seminal study confirmed that even some single neoblasts have the power to rescue the irradiated animal, pro-viding conclusive evidence on the existence of pluripotent adult stem cells (clonogenic neoblasts, cNeoblasts) (Wagner et al., 2011). Finally, although initially considered as homogenous based on morphology, the constantly expanding molecular toolkit, includ-ing FACS and single cell RNA-Seq revealed the heterogeneity of the neoblast population. Currently, several neoblast subclasses representing stem cells and progenitors of dif-ferent lineages are distinguished (Molinaro and Pearson, 2016; Van Wolfswinkel et al., 2014; Wurtzel et al., 2015).

Although the knowledge about the nature of stem cells in planarians has great-ly expanded, a number of questions remain unanswered. For instance, the identification and characterization of the population of truly pluripotent stem cells or elucidating the mechanistic underpinnings of stem cell lineage development would benefit the field enormously (Zhu and Pearson, 2016). Several technical challenges, however, are hin-dering rapid progress. Asexual reproduction significantly limits the use of standard forward genetics approaches, and despite the substantial efforts, transgenic methods for these animals are still not available. It is therefore essential to consider the use of alternative flatworm models, to overcome this major disadvantage.

During the last decade, a promising candidate – Macrostomum lignano has gained attention, as it combines a number of remarkable features identified in planar-ians with the amenability to genetic manipulation (Janicke et al., 2013; Ladurner et al., 2000; Simanov et al., 2012; Wudarski et al., 2017).

Macrostomum lignano

M. lignano (order: Macrostomida) was introduced to the laboratory over a decade ago. Almost immediately, its potential to study stem cell biology, regeneration, development, and sexual selection was recognized (De Mulder et al., 2009; Ladurner, Rieger, & Baguñà, 2000; Ladurner et al., 2005, 2008; Mouton et al., 2009; Nimeth et al., 2004; Schärer & Ladurner, 2003). This free-living marine flatworm is an obligatory non-self-fertilizing hermaphrodite and reproduces exclusively in a sexual manner. The animal is small, about 1 mm long and consists of approximately 25000 cells (Ladurner et al., 2000). Worms are transparent and major tissues and organs can therefore be eas-ily distinguished (i.e. eyes, brain, gonads, reproductive organs, gut) (Figure 2). The sim-plicity of maintaining M. lignano in laboratory conditions is, indisputably, an additional

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advantage. Worms are kept in Petri dishes with nutrient-enriched artificial seawater (f/2) (Anderson et al., 2005), at 20°C and a 14 hr/10 hr light/dark cycle and are fed ad libitum with the diatom Nitzschia curvilineata (Rieger et al., 1988). The combination of a short generation time of about 3 weeks and high fertility rates allows a rapid expan-sion of cultures (Ladurner et al., 2008a).

Figure 2. Bright field image and schematic representation of an adult M. lignano. Scale bar 100 mm.

M. lignano possesses remarkable regeneration potency. Animals are able to re-store missing body parts anteriorly, posteriorly, and laterally, although the presence of the brain and pharynx is obligatory (Egger et al., 2006). These astonishing abilities are attributable to the population of neoblasts. They represent about 6% of all the cells in the adult animal (Bode et al., 2006) and are located in two lateral bands, starting from the region of the eyes and merging in the tail plate. Besides the somatic neoblasts, pro-liferating cells are also present in the gonads (Ladurner et al., 2000). Morphologically, neoblasts are characterized by a small size (5-10µm), round shape and a high nucle-ar:cytoplasmic ratio (Bode et al., 2006). Two molecular markers for somatic neoblasts and germline cells, piwi and vasa, are available and provide an opportunity to further characterize these cell types (De Mulder et al., 2009; Pfister et al., 2007). In contrast to planarians, the heterogeneity of the neoblast population of M. lignano is not yet shown.

brain

eyemouth

testes

ovaries

female opening

egg

stylet

adhesive glands

gut

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1It is worth noting that several molecular techniques were optimized and have

been successfully applied to the animals. These include antibody labelling, in situ hy-bridization (ISH), RNA interference (RNAi), gene expression analysis, and more recently transgenics (Arbore et al., 2015; Ladurner et al., 2000; De Mulder et al., 2009; Pfister et al., 2007; Plusquin et al., 2015; Wudarski et al., 2017). Furthermore, the first genome and transcriptome assemblies became available in 2015 (Wasik et al., 2015) and sub-stantially improved in 2017 (Wudarski et al., 2017). This expanded toolbox allows for in-depth studies of various biological phenomena and in particular in vivo stem cell functioning in connection to mechanisms of regeneration, genome mainte-nance, and ageing, which are discussed in more detail below.

M. lignano as a model for research on stem cells and regeneration

Several aspects of stem cell biology are still poorly understood. For instance, the molecular and cellular underpinnings guiding specialization of stem cells to differ-ent tissues and the mechanism controlling the balance between cell death and cell pro-liferation during homeostasis and regeneration remain fundamental questions in mod-ern biology (Pellettieri and Sánchez Alvarado, 2007; Sánchez Alvarado and Yamanaka, 2014; Zhu and Pearson, 2016). Particularly important is to study these processes in vivo, within the context of a niche and systemic environment, which is very different from the artificial in vitro conditions. Therefore, an accessible in vivo experimental sys-tem is needed.

A common choice for such a model are planarian flatworms. As described ear-lier, a number of advances in understanding certain aspects of neoblast biology, such as knowledge of stem cell heterogeneity and identification of different stem cells subpop-ulations and progenitors, are attributable to studies in planarian flatworms (Molinaro and Pearson, 2016; Van Wolfswinkel et al., 2014; Wurtzel et al., 2015). However, to pro-ceed to more detailed studies of neoblast biology and stem cell biology in general, the need for in vivo cell lineage tracing emerged, which is rather limited in planarians due to the lack of transgenesis and non-transparency.

The recently demonstrated amenability of M. lignano to genetic manipulation (Wudarski et al., 2017) makes this species an exciting complementation to other flat-worm models. In contrast to planarians however, still very little is known about its neo-blast population, and only two stem cell markers, piwi and vasa are available (Ladurner et al., 2008; Pfister et al., 2007, 2008). Hence, before transgenics can be fully appreciat-ed, more detailed characterization of the neoblast population is needed. In addition, as M. lignano is a sexual flatworm (Schärer and Ladurner, 2003), an important distinction

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between proliferating somatic neoblasts and germline has to be made. Therefore, to maximize the potential of M. lignano in stem cell studies, we first generated and char-acterized the transcriptome of proliferating cells in the worm (Chapter 2). This is an essential first step which will, side by side with transgenics, pave the way for further studies of in vivo stem cell functionality

M. lignano as a model for research on stem cells and genome maintenance

DNA damage is one of the main drivers of ageing and cancer, and the conse-quences of DNA damage are particularly severe for adult stem cells (Signer and Mor-rison, 2013). Our understanding of the genome maintenance and its role in stem cell functioning and ageing is steadily improving and is exploited for therapeutic purposes (Pearl et al., 2015). At the same time, further extensive research is required to under-stand the underlying fundamental mechanism in greater detail, and to discover novel therapeutic approaches. Studies focusing on organisms demonstrating exceptional re-sistance to DNA damage, such as tardigrades, are increasingly attractive (Hashimoto et al., 2016). Their powerful DNA protection and DNA damage response system provides novel insights into this important field of research. However, tardigrades do not have a highly proliferative cell compartment (Beltrán-Pardo et al., 2015) and that significantly limits their potential in deciphering genome maintenance mechanisms in stem cells.

To study interplays between DNA damage and stem cell maintenance, an or-ganism that combines an easily accessible stem cell system with high proliferation and that at the same time demonstrates a high resistance to damage by ionizing radiation, is therefore required. This unique combination of characteristics is present in M. lignano (De Mulder et al., 2010; Ladurner et al., 2008). To explore its potential as an experimen-tal organism for research on stem cells and genome maintenance, we characterized the early transcriptional response of the worm to the ionizing irradiation (Chapter 5).

M. lignano as a model for research on stem cells and ageing

A progressive deterioration of homeostatic and regenerative capacities of age-ing tissues is known to be driven by dysregulation of proliferative activities and a de-cline in stem cell functioning (Rando, 2006). That in turn, is often attributable to the cellular damage that is accumulated by the stem cell pool in the course of the lifetime and/or to age-imposed biochemical alterations in the niches housing stem cells. Indeed, several causes can result in a decreased stem cell functionality, an integrative hallmark of ageing which will inevitably lead to age-related diseases and eventually death (Lo-pez-Otin et al., 2013; Rando, 2006; Sharpless and DePinho, 2007). In consequence, an

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1exciting possibility of slowing down or even reversing the ageing of stem cells and their niches, could potentially lead to deceleration of ageing of the entire organism. In this regard, an evidence derived from experiments involving heterochronic parabiosis, in-dicated that providing the cellular microenvironment derived from young animals leads to enhanced functioning of the aged stem cell pool (Conboy and Rando, 2005, 2012; Conboy et al., 2005; Loffredo et al., 2013).

Interestingly, the stem cell ‘rejuvenation’ hypothesis was also explored in flat-worms. Several experiments suggested that animals are able to attenuate or even re-verse the ageing process, through repeated regeneration, thereby ‘rejuvenating’ the stem cell pool (Child, 1911, 1915; Hyman, 1951). The most recent reports, indicates that several amputations in M. lignano significantly extend its lifespan compared to in-tact control animals (Egger, 2008; Egger et al., 2006). This initial evidence makes M. lig-nano a tractable model for research on stem cells and ageing, which is further explored in Chapter 4.

M. lignano as a model for research on flatworm-specific biology

Studying flatworm-specific biology carries interesting biomedical potential. It was shown before that research using free-living animals provided meaningful insight into knowledge about their parasitic relatives (Foster et al., 2005; McCarter, 2004). For instance, a recent study in the planarian Schmidtea mediterranea, dissecting the mech-anisms of early planarian male germ cells maintenance, demonstrated that the find-ings derived from the free-living species were also applicable to the parasitic flatworm, Schistosoma mansoni (Iyer et al., 2016). This has important implications for exploring treatment targets for infections caused by parasitic agents.

Here we propose, that certain characteristics of M. lignano, such as the repro-duction in a sexual manner, the ease of culture and access to the screening tools (in situ hybridization, RNAi, transgenics) creates a convenient experimental platform to study flatworm features, and in consequence, allows to extend the findings to parasitic flat-worm species. In Chapter 3 we present a proof-of-principle study for the functional characterization of nonconserved genes.

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Thesis outline

M. lignano is poised to become a powerful invertebrate model organism for stem cell research once its experimental toolbox is well-established and sufficient knowledge on basic understanding of its biology is accumulated. Since M. lignano is still a very young a model, the current work in our laboratory focuses on developing tools and resources for this animal and on investigating various aspects of its stem cell biology. After all, it is true that “[…] an initial phase to establish a core set of experimen-tal protocols is necessary to attract a critical mass of researches, a prerequisite for the long-term success of a model organism. The greater the accumulated body of knowledge, the easier it is to address biological questions by experiments without being tied down by technical obstacles” (Müller & Grossniklaus, 2010). This thesis summarizes the efforts to advanced M. lignano as a versatile model organism and provides the evidence that M. lignano is a suitable platform to study a number of different biological phenomena.

Chapter 1 provides an overview of traditional and nontraditional model or-ganisms popular in modern research. The chapter emphasizes that some of the intrigu-ing questions in biology cannot be answered using ‘standard’ models, leading to the need of alternative animal systems.

In Chapter 2, a de novo transcriptome assembly for M. lignano is presented. Furthermore, the chapter offers a comprehensive characterization of gene expression in the somatic stem cells and proliferating germline cells of the worm. These transcrip-tional signatures were further used to identify novel markers for somatic neoblasts. Finally, a user-friendly interface was designed, which provides access to the generated data and, potentially, will facilitate the use of the animal in stem cell biology research.

Chapter 3 scrutinizes the value of nonconserved genes and explores its poten-tial in biomedical research. It also presents the experimental screening platform for ef-ficient characterization of the expression pattern and function of potentially interesting gene candidates.

Chapter 4 explores advantages of M. lignano as a model organism for ageing research. Several ageing biomarkers and transcriptional signatures of ageing are pre-sented. Moreover, a long-standing hypothesis of rejuvenation is tested.

Chapter 5 provides a transcriptomic insight into the remarkable resistance of M. lignano to γ-ray radiation and describes the initial efforts to optimize a comet assay to quantify the DNA damage in the cells of the worm.

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1Finally, Chapter 6 gives a summary of the work described in this thesis and the

major implications of these results to the scientific community are discussed. Further-more, the future directions are explored.

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