University of Groningen Development of genetic manipulation tools in Macrostomum lignano for dissection of molecular mechanisms of regeneration Wudarski, Jakub

University of Groningen

Development of genetic manipulation tools in Macrostomum lignano for dissection of

molecular mechanisms of regeneration

Wudarski, Jakub

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Development of genetic

manipulation tools in

Macrostomum lignano for dissection

of molecular mechanisms of

regeneration

ISBN (printed version): 978-94-034-1318-1

ISBN (electronic version): 978-94-034-1317-4

Development of genetic manipulation

tools in Macrostomum lignano for

dissection of molecular mechanisms of

regeneration

PhD thesis

to obtain the degree of PhD at the University of Groningen

on the authority of the Rector Magnificus Prof. E. Sterken

and in accordance with the decision by the College of Deans. This thesis will be defended in public on Monday 14 January 2019 at 12.45 hours

by

Jakub Wudarski

born on 8 January 1986 in Torun, Poland

Supervisor

Prof. E. Berezikov Co-supervisor Prof. V. Guryev

Assessment Committee Prof. E.A.A. Nollen

Prof. F.M. Reggiori Prof. M. Tijsterman

To my family

Mojej Rodzince

CONTENTS

CHAPTER

1

9

General introduction and the outline of the thesis

CHAPTER

2

15

The regenerative flatworm Macrostomum lignano, a model organism with

high experimental potential

CHAPTER

3

33

Efficient transgenesis and annotated genome sequence of the regenerative flatworm model Macrostomum lignano

CHAPTER

4

71

Proof of principle for transposon-mediated transgenesis in

Macrostomum lignano

CHAPTER

5

85

Influence of temperature on the development, reproduction and regeneration in the flatworm model organism Macrostomum lignano

CHAPTER

6

103

Flourescentce activated cell sorting is an efficient approach for establishing transcriptional profiles of various tissues in the flatworm

Macrostomum lignano

CHAPTER

7

127

Summary and Discussion Future perspectives

APPENDICES

133

CHAPTER 1

General introduction and the

outline of the thesis

OPENING REMARKS

Influencing the genomes of various organisms has been present in the human history for thousands of years. Current standing hypothesis considers the domestication of the gray wolf dating back to around 12000 BC as the first event when humans had influenced the process of genetical change via artificial selection [1]. With the development of our civilization humans were able to affect the surrounding biology in more various and sophisticated ways including selective breeding, hybridization and grafting [2]. The breakthrough came with the onset of modern genetics. The discovery of restriction enzymes in 1970 [3] started the genetic engineering as we know this term today. In other words, people finally started to understand what they were doing in terms of genetic manipulation. Already in 1973, the first genetically modified bacteria were created [4] and only a year later the first transgenic mouse was born [5]. With almost every major discovery in the field of molecular biology, transgenic techniques were improving. The use of, already known, transposable elements, discovery of polymerase chain reaction (PCR), fluorescent proteins, Transcription Activator-Like Effector Nucleases (TALENs) or the most recent breakthrough – Clustered Interspaced Palindromic Repeats/ Cas9 (CRISPR/Cas9), all pushed the boundaries of what scientists could decipher about the biology of different organisms [6–11]. Throughout the years genetic manipulation rose to one of the hallmarks of the scientific progress. Present in the popular culture, movies, comic books, sci-fi novels, modifying genomes represents one of humanities great achievements. The reality, however, tends to be a lot different than what is presented in the cinemas. Today, after almost fifty years, despite the magnificent progress that was achieved in the field of transgenics, we still have plenty of blank spots that can be filled in, some of which requiring a proper approach rather then a scientific brakethrough.

One of such blank spots is the genetic manipulation in flatworms. Flatworms have been known for their regenerative capabilities for over a century [12], and they are a popular choice when it comes to model organisms. Their unique ability comes from a population of adult stem cells referred to as neoblasts [13, 14]. One can imagine that in-depth characterization of neoblasts should improve our understanding of processes such as regeneration and stem cell differentiation. There has been a lot of effort put into deciphering the functioning of these cells, proving their pluripotency [15] or showing that the population is, in fact, heterogeneous [16]. Despite the progress we are still unable to reliably isolate or tag the neoblasts. One of the reasons for this is the lack of available transgenic techniques necessary to efficiently modify the flatworm genome. This also severely hampers our understanding of the biology of flatworms, currently making them a less attractive model for any genetic studies.

This thesis is focused on Macrostomum lignano, a marine free-living flatworm with high

regenerative potential. The overall aim of this work was to improve on the currently available techniques for flatworm genetic studies by developing a molecular toolkit for genetic manipulation and provide sufficient evidence to make M. lignano is a versatile and robust

THESIS OUTLINE

Chapter 2 introduces Macrostomum lignano as a model organism giving an overview on

the biology of the animal. It also explores the potential research areas that could benefit from using the flatworm as either substitute or supplementary model organism to answer the questions related to regeneration and stem cell biology.

Chapter 3 presents the development and implementation of the transgenic techniques

for Macrostomum lignano. It introduces the molecular toolkit for genetic manipulation in

M. lignano. The toolkit is focused on the microinjection approach, which is shown to be robust,

reproducible and reliable. Up to today it is the only available method to create stable transgenic flatworms. This chapter also presents the most up-to-date M. lignano genome assembly together

with the updated transcriptome assembly and genome annotation. The molecular toolkit from this chapter will be further expanded and tested in throughout the thesis.

Chapter 4 explores the use of the piggyBac transposon system to facilitate stable integration

of the desired DNA fragments. It focuses on advantages that this approach offers as compared to the method presented in Chapter 3. It also highlights new directions in which further development of the genetic manipulation techniques for M. lignano can go.

Chapter 5 demonstrates the influence of the temperature on the basic aspects of the flatworm biology, including development, reproduction and regeneration. It also presents how transgenic animals, made using the techniques described in chapter 3 are used for research purposes. Finally, it shows how temperature manipulation can facilitate future experiments that will be performed using Macrostomum lignano.

Chapter 6 is focused on how transgenic lines expressing GFP under tissue-specific promoters can be used to isolate desired cell populations by FACS and characterize them using RNA-seq. The specificity of the technique enables more detailed characterization of flatworm neoblasts by more precisely separating them from the germline. This chapter presents practical application of the available transgenic lines. It also highlights how data obtained using M. lignano can supplement current findings and point the way for future

research.

Chapter 7 summarizes the findings presented in this thesis and gives a brief overview of the obtained results. It also explores their relevance and potential impact on future research.

REFERENCES

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[5] R. Jaenisch and B. Mintz, “Simian Virus 40 DNA Sequences in DNA of Healthy Adult Mice Derived from Preimplantation Blastocysts Injected with Viral DNA,” Proc. Natl. Acad. Sci., vol. 71, no. 4, pp. 1250–1254, 1974.

[6] A. P. D. Chalfie M, Tu Y, Euskirchen G, Ward WW, “Green fluorescent protein as a marker for gene expression,” Science (80-. )., vol. 263, no. February, pp. 802–805, 1994. [7] B. McClintock, “Induction of instability at selected loci in maize,” Genetics, vol. 38, no. 6, p. 579 LP-599, Nov. 1953.

[8] K. Mullis, F. Faloona, S. Scharf, R. K. Saiki, G. T. Horn, and H. Erlich, “Specific enzymatic amplification of DNA in vitro: the polymerase chain reaction,” in Cold Spring Harbor symposia on quantitative biology, 1986, vol. 51, pp. 263–273.

[9] M. Jinek, K. Chylinski, I. Fonfara, M. Hauer, J. A. Doudna, and E. Charpentier, “A Programmable Dual-RNA–Guided DNA Endonuclease in Adaptive Bacterial

Immunity,” Science (80-. )., Jun. 2012.

[10] N. Kleckner, J. Roth, and D. Botstein, “Genetic engineering in Vivo using translocatable drug-resistance elements: New methods in bacterial genetics,” J. Mol. Biol., vol. 116, no. 1, pp. 125–159, Oct. 1977.

[11] A. J. Wood, T.-W. Lo, B. Zeitler, C. S. Pickle, E. J. Ralston, A. H. Lee, R. Amora, J. C. Miller, E. Leung, X. Meng, L. Zhang, E. J. Rebar, P. D. Gregory, F. D. Urnov, and B. J. Meyer, “Targeted genome editing across species using ZFNs and TALENs.,” Science, vol. 333, no. 6040, p. 307, 2011.

[12] T. H. Morgan, “Experimental studies of the regeneration of Planaria maculata,” Arch. fur Entwickelungsmechanik der Org., vol. 7, no. 2–3, pp. 364–397, 1898.

[13] J. Baguña, “The planarian neoblast: the rambling history of its origin and some current black boxes,” Int. J. Dev. Biol., vol. 56, no. 1-2–3, pp. 19–37, 2012.

[14] J. C. Rink, “Stem cell systems and regeneration in planaria,” Development Genes and Evolution. 2013.

[15] D. E. Wagner, I. E. Wang, and P. W. Reddien, “Clonogenic neoblasts are

no. 6031, pp. 811–816, 2011.

[16] J. C. van Wolfswinkel, D. E. Wagner, and P. W. Reddien, “Single-cell analysis reveals functionally distinct classes within the planarian stem cell compartment.,” Cell Stem Cell, vol. 15, no. 3, pp. 326–39, Sep. 2014.

CHAPTER 2

The regenerative flatworm

Macrostomum lignano, a model

organism with high experimental

potential

Stijn Mouton†,1_{, Jakub Wudarski}†,1_{, Magda Grudniewska}1,2 _{and Eugene} Berezikov*,1

1_{European Research Institute for the Biology of Ageing, University of Groningen, University Medical Center Groningen,} Groningen, The Netherlands.

2_{Present address: Center for Neurodegenerative and Neuroimmunologic Diseases, Department of Neurology, Rutgers – Robert Wood} Johnson Medical School, Piscataway, USA.

†_{These authors contributed equally to this work.} *_{Corresponding author.}

ABSTRACT

Understanding the process of regeneration has been one of the longstanding scientific aims, from a fundamental biological perspective as well as within the applied context of regenerative medicine. Because the regeneration competence varies greatly between organisms, it is essential to investigate different experimental animals. The free-living marine flatworm Macrostomum lignano is a rising model organism for this type of research,

and its power stems from a unique set of biological properties combined with amenability to experimental manipulation. The biological properties of interest include production of single-cell fertilized eggs, a transparent body, small size, short generation time, ease of culture, the presence of a pluripotent stem cell population, and a large regeneration competence. These features sparked the development of molecular tools and resources for this animal, including high-quality genome and transcriptome assemblies, gene knockdown, in situ hybridization,

and transgenesis. Importantly, M. lignano is currently the only flatworm species for which

transgenesis methods are established. This review summarizes biological features of M. lignano

and recent technological advances towards experimentation with this animal. In addition, we discuss the experimental potential of this model organism for different research questions related to regeneration and stem cell biology.

INTRODUCTION

Regeneration of missing or damaged body parts and tissues is an intricate multicellular process. The ability to regenerate is broadly present within the Metazoa but its extent shows large variation [1–4]. Animals like Hydra can regenerate the whole body [5], while

in human regeneration is limited to certain tissues and organs, such as the liver [6]. Despite the longstanding fascination of scientists for regeneration and the promise of enhancing human tissue and organ repair by regenerative medicine, the regenerative process is still poorly understood [7]. To advance developments in regenerative medicine, it is important to study regeneration in an evolutionary context and to increase our understanding of the basic biology underlying tissue repair [7]. Therefore, scientists study the molecular and cellular mechanisms of regeneration in a wide variety of experimental animals, such as Hydra,

flatworms, crustaceans, amphibians and zebrafish [2, 3].

In this concise review we focus on the flatworm Macrostomum lignano. As a representative

of the Macrostomorpha, M. lignano is a basal member of the flatworms (Platyhelminthes),

while the well-known models Schmidtea mediterranea and Dugesia japonica are members of the

more derived clade of Tricladida (Fig. 1A). M. lignano was first brought into the laboratory in

1995 [8], and has since been used as a model for stem cell biology, development, regeneration, ageing, sex allocation, sperm competition, and bio-adhesion [9–18]. During the last years, important progress was made in the development of molecular tools for this animal, such as the establishment of transcriptome and genome assemblies, and the development of transgenic methods [19–21]. Here, we describe the key aspects of the biology of M. lignano, which make it

a convenient model for experimentation, discuss the recent progress of developing molecular tools and resources, and illustrate the potential of this model for research areas related to regeneration and stem cell biology.

THE BIOLOGY OF

MACROSTOMUM LIGNANO

The flatworm M. lignano is a free-living, marine species. Its natural environment is the

high-tide interstitial sand fauna of beaches of the Northern Adriatic Sea, Italy [8, 22]. In the laboratory, the worms can be easily cultured in Petri dishes with nutrient-enriched artificial sea water (f/2) at a salinity of 32 ‰. The dishes are maintained at 20˚C and a 14h/10h or 13h/11h light/dark cycle, and worms are fed ad libitum with the diatom Nitzschia curvilineata,

which is grown in the same conditions [23].

M. lignano is an obligatory non-self-fertilizing hermaphrodite, which reproduces

exclusively in a sexual manner [15]. Mass cultures can be quickly established, as adult worms lay about one egg each day at 20˚C. The embryonic development lasts 5 days, while the total generation time is about 18 days. In contrast to planarians, which exhibit a highly derived embryogenesis, sometimes referred to as “blastomere anarchy” [24, 25], the early embryonic cleavages of M. lignano are of the quartet spiral type, while in the later stages the process of

“inverse epiboly” takes place, meaning that the blastomeres at the vegetal pole form an outer yolk mantle at the surface of the embryo [10]. It is important to note that the eggs follow the entolecithal (archoophoran) mode of development (Fig. 1A) [26, 27], making them an easy target for microinjections, as the single cell has a relatively large size of 100 µm (Fig. 1B).

The morphology of M. lignano is described in detail [8, 28]. Defining characteristics are

its small size (about 1.5 mm), the presence of two pigment-cup eyes enabling light sensing, and the transparency of the complete body. Internal organs, such as the brain, gut, testes, ovaries, and developing eggs can be easily observed (Fig. 1B). The tail plate with a slightly curved stylet, representing the male reproductive organ, and adhesive glands, enabling the animal to attach and detach from most surfaces, is another morphological characteristic of

Macrostomum [29].

The regenerative flatworms are usually perceived through planarians, which have almost unlimited regenerative capabilities. The Macrostomorpha are, however, not far behind and also have an excellent regeneration capacity when compared to most of the free-living flatworm taxa [30]. Figure 1C illustrates how an amputated head of M. lignano can regenerate

the complete body, including the germline, within 3 weeks. M. lignano is able to regenerate

missing body parts anteriorly, posteriorly, and laterally, although the presence of the region proximal to the brain, eyes and pharynx is obligatory. In other words, when cut in half, only the head-piece will regenerate and survive [13]. Interestingly, in planarian species which have limited capacity of head regeneration, the regeneration deficiency can be rescued by downregulation of canonical Wnt signalling [31, 32]. Similarly, although the amputated tail region if M. lignano cannot regenerate the head under normal conditions, there is anecdotal

evidence that head regeneration can be induced [33], but further research is needed to fully unravel these mechanisms. The regenerative capacity after different types of amputation is described in detail by Egger et al. [13]. Interestingly, a limit of the regeneration competence of this species has not been fully identified yet. Even after 29 consecutive amputations of the body, a decline of the regeneration rate could not be observed [13]. Moreover, there are reports of individuals surviving almost 60 consecutive amputations [13, 34].

brain eyes mouth testes ovaries female opening egg stylet adhesive glands gut B C 3 weeks Catenulida Macrostomorpha Polycladida Prorhynchida Gnosonesimida Rhabdocoela Proseriata Fecampiida Prolecithophora Tricladida Bothrioplanida Cestoda Monogenea Digenea SP SP SP SP SP EN EN EN EC EC EC EC EC EC EC EC BA BA A Neodermata

Fig. 1. Phylogeny, morphology and regeneration of Macrostomum lignano. (A) Schematic representation of the phylogeny of the flatworms (Platyhelminthes). The tree and the presence of spiral cleavage (SP) is based on Laumer et al., 2015. The presence of entolecithal (EN) or ectolecithal (EC) eggs, and blastomere anarchy (BA) is based on Egger et al., 2015. (B) Bright field image and schematic representation of an adult worm. Scale bar: 100 µm. (C) Regeneration cycle from a head fragment into an adult, fertile worm, of which the body can be amputated again.

At the cellular level, the body of M. lignano can be described as a dynamic steady state. In

total, there are about 25.000 cells and there is a high cellular turnover during homeostasis [22, 35]. The epidermis is an interesting example, as about one third of all cells are renewed within two weeks [35]. As in all flatworms, the source of new cells during regeneration and homeostasis are stem cells called neoblasts (Fig. 2A,B), which are the only somatic proliferating cells in the adult body [22]. Importantly, besides the somatic neoblasts, proliferating cells are also present in the gonads [22] (Fig. 2A). The neoblasts are morphologically characterized as small, round cells (6-10 µm) with a high nuclear:cytoplasmic ratio [36]. The large nucleus has a prominent nucleolus, and the cytoplasm has few organelles limited to free ribosomes, few mitochondria, and none or very little endoplasmic reticulum [22, 35, 36] (Fig. 2B). A comprehensive summary of our knowledge about the flatworm neoblast population is provided in reviews by Baguña [37] and Rink [38]. In M. lignano, the neoblasts are located

within the parenchyma, in two bands along the lateral sides of the animal, merging in the tail plate. The typical pattern of the neoblasts can be explained by the close relation of their location to the main lateral nerve chords [9] (Fig. 2A). In the rostrum, the region in front of the eyes, neoblasts are absent [22] (Fig. 2A). It is estimated that about 6.5% of all cells are somatic neoblasts, of which 27% are in S-phase [22, 36, 39]. The neoblast population is assumed to be actively proliferating, but there are indications that a small fraction of the population is slow cycling or quiescent [36, 40]. The commonly used methods to visualize the neoblasts is to label cells in S-phase by means of BrdU incorporation, or to label the mitotic cells with a polyclonal antibody against phosphorylated Histone H3 [9, 22]. In addition, in situ hybridization can be performed to visualize the expression of piwi and vasa, which are

expressed in all proliferating cells, representing both somatic neoblasts and germline cells [41–43]. To isolate proliferating cells, a cell sorting approach based on the 4C DNA content of Hoechst-labeled cells in the late S-, G2, and M-phase of the cell cycle is used [19]. The somatic neoblast population as a whole is pluripotent and able to form all cell types present in the body, including the germline. However, based on research in freshwater planarians [44–46], it is assumed that the neoblasts in M. lignano represent a heterogeneous population

of pluripotent stem cells and subsets of stem cells and progenitors of different differentiation lineages. However, direct data about the heterogeneity of neoblasts in M. lignano is currently

lacking, and more research is needed to characterize the different types of neoblasts, unravel differentiation lineages, and develop better markers for different types of somatic neoblasts. The recently developed transgenesis methods represent a convenient tool for such studies and will be discussed below.

A B

EP

Fig. 2. | The neoblast population of Macrostomum lignano. (A) Confocal projection of BrdU (green: S-phase cells) and phospho-histone H3 (red: mitotic cells) immunostaining of an adult worm. Note the bilateral pattern of the proliferating cells. (B) Nanotomy of M. lignano [19]. Top to bottom represents zooming in on the boxed areas, starting with a complete cross section and finishing with a detailed view illustrating the ultrastructure of a neoblast and its surrounding. The nucleus of the neoblast (orange) is characterized by patchy clumps of condensed chromatin and a large nucleolus, and is surrounded by a thin rim of cytoplasm (red). The neoblast lays in direct contact with the lateral nerve chord (green) and muscle tissue (blue). EP indicates the epidermis of the animal. Scale bars from top to bottom: 25 µm, 10 µm, 1 µm.

GENOMIC TOOLS AND RESOURCES

Whenever faced with choosing a suitable model organism for a particular research question, scientists look at the available techniques that are established for that model. These techniques should be specific and relatively easy to perform. In case of M. lignano, in situ

hybridization (ISH) and RNA interference (RNAi) are well established and widely used in the community. ISH uses a standard protocol, previously established in planarians and is a powerful method for showing expression patterns of selected genes, although it is quite laborious and suboptimal for genes with low expression [41]. RNAi is performed by simple soaking of the animals in double stranded RNA solution [43]. It is relatively easy to do but requires changing the solution on a daily basis. Both methods have a rather low throughput

but can be used as screening methods and are essential techniques for a wide range of research questions in M. lignano [42, 47]. However, it is the simple and robust method for generating

transgenic animals that makes M. lignano to stand out and gives it a substantial advantage over

planarian model organisms. The worms lay relatively large (100 microns in diameter), single-cell fertilized eggs that can be easily picked using plastic pickers and are a perfect material for microinjections. These eggs provide a very convenient route for delivering biologically active materials, such as RNA, DNA, proteins or chemicals directly into the organism. Recent results show that injecting simple double stranded DNA fragments can result in their integration into the genome and the establishment of stable transgenic lines that can express fluorescent protein in the tissues of interest [21]. Because of the transparency of the worm, these marker proteins can be easily visualized by the use of light-, fluorescent-light or confocal microscopy on both fixed or live samples (Fig. 3) [21, 29]. Additionally, thanks to the extensive efforts of several research groups, a wealth of electron microscopy data exists (Fig. 2B) that provides a reliable source of morphological information for anyone willing to start working with these flatworms [36, 48–50]. Even though to date there are no reports about the use of more controllable genome manipulation technologies, such as transposons, integrases or the CRISPR/Cas9 system, based on the results on random integration of microinjected DNA one might expect these methods to be developed in the near future.

In the era of advanced molecular biology, having a sequenced and annotated genome is one of the key features a model organism must have. Sequencing of the genome of M. lignano was

one of the key projects initiated by the Macrostomum community, which resulted in an early

assembly based on PacBio sequencing data [20], and later on was substantially improved by the addition of 454 and Illumina sequencing data and the use of the latest genome assembly algorithms [51]. The size of the current M. lignano genome assembly Mlig_3_7_DV1, which

is based on the widely used DV1 line, is 760 MB, and corresponds to the experimentally measured genome size for this line. The N50 contig and scaffold sizes of the assembly are 215.2 Kb and 245.9 Kb respectively [21]. While further improvement of the genome assembly continuity is desirable, the current version already provides sufficient information for the majority of research questions.

To make full use of a genome assembly, it requires a detailed gene and repeat annotation. Therefore, substantial efforts were dedicated to annotate the genome using a large set of RNA-seq data [19, 21, 50]. The resulting genome-guided transcriptome assembly Mlig_RNA_3_7_ DV1 is 98.1% complete as measured by the Benchmarking Universal Single-Copy Orthologs [52], with only 3 missing and 3 fragmented genes. It incorporates the expression data from RNA-seq libraries generated using various approaches, such as strand-specific polyA-enriched and RiboMinus-depleted Illumina libraries and 5'-enriched RAMPAGE libraries, which can provide important additional information regarding expression levels, transcription start sites, transcription terminators and trans-splicing [21]. All of the abovementioned data are visualized using UCSC genome browser software and are publicly available

(http://gb.macgenome.org). Additionally, a database of genes enriched in the proliferating neoblasts and germline cells are also available in the open access form (http://neoblast. macgenome.org). These resources were used to select several tissue-specific genes and promoters and proved to be highly reliable for ISH, RNAi and transgenic experiments [19, 21, 50].

A

B

Fig. 3. Examples of tissue-specific transgenes in M. lignano. (A) Transgenic M. lignano animal from line NL24 [21], where ELAV promoter drives the expression of mNeonGreen in the testes, and spermatozoa located in the vas deferens and seminal vesicle (shown in green), and CABP7 promoter drives the expression of mScarlet in ovaries and developing eggs (shown in red). (B) 3D visualization of confocal Z-stack image of the tail of a trans-genic worm from line NL9 [21] expressing GFP under the muscle specific MYH promoter.

RESEARCH QUESTIONS

The availability of a broad set of genomic tools and resources provides a solid base for further detailed characterization of M. lignano, and gives the necessary means to address some of the

pressing biological questions. M. lignano is becoming a versatile model organism and can be

used in various research fields as main or supplementary choice. Here we focus on several research directions, where, in our opinion, M. lignano can be a particularly suitable model.

Stem cells and regeneration

The most prominent and best recognizable field of research connected to flatworms is the one regarding stem cells and regeneration. As mentioned before, the source of the astonishing regeneration capabilities of flatworms is a population of stem cells called neoblasts. These cells have been, and still are, extensively studied by various research groups. In 2011 the definitive proof of neoblast pluripotency has been demonstrated. In an elegant experiment using single cell transplantation in Schmidtea mediterranea, scientists showed that a single

neoblast can restitute the regeneration ability of an otherwise regeneration incapable worm [53]. Additional studies using this model provided the first insight in the embryonic origin of neoblasts [54] and the heterogeneity of the adult neoblast population, by means of single cell transcriptomics [45, 46]. Despite this progress, we are still far from fully understanding the biology of these fascinating cells. In a recent attempt to characterize neoblasts more accurately in M. lignano, Grudniewska and Mouton et al. [19] performed a molecular characterization of

all of the proliferating cells in the worm. This was achieved by generating two transcriptomes of proliferating cells: the first one based on worms devoid of neoblasts through irradiation, and the second approach based on FACS selection. As a result, a list of neoblast stringent candidate genes was composed, which can help to overcome one of the major problems that still persists in the field: the lack of efficient tagging and tracking of neoblasts. This is also the point where the main advantage of M. lignano becomes most relevant. Having efficient ways

for generating transgenic animals and, with the advent of CRISPR/Cas9 technology, tagging neoblasts or using the FUCCI system [55, 56] to track their fate during regeneration would be a milestone in the process of characterizing different cell lineages and understanding the details of the differentiation of these cells.

Ageing and rejuvenation

The concept of tissue rejuvenation during regeneration, and the consequent potential immortality are long-standing hypotheses in the field of flatworm biology [34, 57–60].

M. lignano represents a fascinating model to study connections between stem cells, ageing

and regeneration [14]. In contrast to asexual planarians, which reproduce by fission and are considered immortal, and therefore have their own value for ageing research [60, 61],

M. lignano does age, albeit much slower than e.g. C. elegans or flies [14, 62].

morphology, fertility, and gene expression were studied as a function of age in 3 regenerative conditions: intact worms, worms which regenerated their body once, and worms which regenerated their body multiple times [63]. Interestingly, the used inbred DV1 strain appeared to live substantially longer than the previously used wild type strain. Although phenotypic signs of ageing were observed, regenerative abilities were not decreased with advancing age, even in 26 months old animals that had their body amputated and regenerated repeatedly for 12 times. At the same time, the study demonstrated that single and multiple whole-body regeneration does not result in rejuvenation of M. lignano [63], supporting the hypothesis

that rejuvenation might be limited to asexual species [59]. The detailed analysis of the RNA sequencing data revealed significant upregulation for somatic neoblast transcripts in old animals, including genes known to be essential for neoblast functionality, such as PIWIL1, DDX39B, CDK1, RRM1, and H2AFV [19]. In addition, several genes with known beneficial effects on lifespan when overexpressed in mice and C. elegans are naturally upregulated with

age in M. lignano. Taken together, the gene expression data suggest that M. lignano evolved

molecular mechanisms to offset negative consequences of ageing [63]. It will be interesting to further study M. lignano genes with age-specific expression patterns e. g. by means of genetic

engineering in the context of other model organisms, which represents a promising research direction to explore anti-ageing strategies [64].

Genome maintenance and chromosome evolution

Precise and faithful replication, preservation and damage repair of DNA are the key points in the maintenance of the genome of an organism. Any source of damage, such as polymerase errors, radiation, chemicals, pH, temperature, or any other stress can push the system off balance and become fatal to the cell. Fortunately, cells have many ways to deal with stress conditions, such as DNA damage response and heat shock response pathways, that help keeping their key molecules intact. Studying these mechanisms is important not only from general biological perspective, but can also lead to major advancements in fields like cancer biology and medicine [65, 66]. Cancer research is one of the most highly financed branches of science but despite the enormous efforts put into deciphering the whole process of cancer, we are still unable to fully characterize it [67, 68]. Like with most fields in science, choosing an appropriate model organism is dictated by the type of biological question and the characteristics of the potential model. To date, there have been no reports on naturally occurring cancer in M. lignano, despite its high cellular proliferation rate. The worm is also

highly resistant to external sources of DNA damage, such as ionizing radiation, being able to survive up to 210 Gy of γ-radiation, compared to just a few Gy in mammals [19, 69]. Efficient DNA protection and/or repair machinery must underlie this remarkable resistance of the animal to DNA damage, and investigating its molecular mechanisms holds a great promise.

Furthermore, it was shown recently that several species of Macrostomum undergo

potential of M. lignano as a model organism for research on genome and chromosome evolution,

genome maintenance and cancer.

Flatworm-specific biology

Flatworms are an object of scientific research for more than a hundred years. The first experiments were done on planarians and documented by Harriet Randolph and Thomas H. Morgan [72, 73]. Platyhelminthes are definitely a great and appealing model when studying regeneration. It is not, however, the only field where understanding flatworm biology can be important and beneficial. For example, in the case of Macrostomum we can see a very special

ability of adhering to various surfaces [29]. This ability is even more astonishing when we consider the fact that the worms can not only attach but also detach at will, which is a very rare ability in the animal kingdom, and a detailed understanding of this process might help us advance even the field of engineering. Another example is the research performed

on Neodermata, a taxa of obligatory parasitic flatworms, such as tapeworms and blood

flukes, which are one of the major focuses of health-related studies performed in flatworms. However, difficulties in culturing these parasites strongly hamper the progress in the field. Having a free-living flatworm model which is easy-to-work with can substantially speed up the progress in the field of parasitic flatworm biology. M. lignano is a suitable model for these

studies, with the availability of transgenic techniques as the major point in its favor.

To identify therapeutic gene targets without side-effects on human biology, an attractive approach is to focus on genes conserved in flatworms but not in human. Moreover, studying flatworm-specific genes is essential for understanding flatworm biology. While molecular flatworm research traditionally focuses on broadly conserved genes, including conservation in human, two recent papers illustrated the importance of the flatworm-specific approach. The first study, describing gene expression during tail regeneration, identified three novel previously unannotated Mlig-stylet genes, which are required for the formation of the male copulatory apparatus [74]. The second study focused on a previously unannotated flatworm-specific gene Mlig-sperm1, which is part of a novel gene family, and is flatworm-specifically expressed in the testes and required for producing healthy spermatozoa, and consequently, the fertility of the worms [50]. In conclusion, Macrostomum research is demonstrating the importance of

studying flatworm-specific biology. This novel approach has the potential to impact different fundamental, but also applied research fields.

CONCLUDING REMARKS

Free-living flatworms are mainly known for their regenerative capacity and represent powerful models for a broad range of research questions related to stem cell biology and regeneration. While being a rather novel flatworm model, the species Macrostomum lignano has

high experimental potential due to a combination of interesting and convenient biological properties and the recent progress in developing genomic tools and resources, including

transgenesis (Fig. 4). The availability of transgenesis methods in particular might make

M. lignano the future flatworm model of choice for in depth molecular studies on stem cells

and regeneration, but also expand its use into other fields of research, such as cancer, genome maintenance and ageing.

Genome

Transcriptome

Transgenesis

RNA interference

Stem cell biology Regeneration

Genome maintenance Chromosome evolution Flatworm-specific biology Ageing TOOLBOX RESEARCH AREAS In situ hybridization FACS/Flow Cytometry Nanotomy

Fig. 4. | Experimental potential of Macrostomum lignano. Summary of experimental tools and resources avail-able for M. lignano and some of the research areas where this model has large potential.

ACKNOWLEDGEMENTS

This work was supported by the European Research Council (ERC Starting Grant “MacModel”, grant no. 310765) to E.B.

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CHAPTER 3

Efficient transgenesis and annotated

genome sequence of the regenerative

flatworm model

_{Macrostomum lignano}

Jakub Wudarski1_{, Daniil Simanov}1,2_{, Kirill Ustyantsev}3_{, Katrien de Mulder}2,8_, Margriet Grelling1_{, Magda Grudniewska}1_{, Frank Beltman}1_{, Lisa Glazenburg}1_, Turan Demircan2,10_{, Julia Wunderer}4_{, Weihong Qi}5_{, Dita B. Vizoso}6_{, Philipp M.} Weissert1_{, Daniel Olivieri}1,9_{, Stijn Mouton}1_{, Victor Guryev}1_{, Aziz Aboobaker}7_, Lukas Schärer6_{, Peter Ladurner}4_{, Eugene Berezikov}1,2,3

1_{European Research Institute for the Biology of Ageing, University of Groningen, University Medical Center Groningen,} Groningen, The Netherlands.

2 Hubrecht Institute-KNAW and University Medical Centre Utrecht, Uppsalalaan 8, 3584CT, Utrecht, The Netherlands. 3 Institute of Cytology and Genetics, Prospekt Lavrentyeva 10, 630090 Novosibirsk, Russia.

4 Institute of Zoology and Center for Molecular Biosciences Innsbruck, University of Innsbruck , Technikerstr. 25, A-6020, Innsbruck, Austria.

5 Functional Genomics Center Zurich, Winterthurerstrasse 190, Zurich, CH-8057, Switzerland. 6 Evolutionary Biology, Zoological Institute, University of Basel, Vesalgasse 1, CH-4051, Basel, Switzerland.

7 Department of Zoology, University of Oxford, Tinbergen Building, South Parks Road, Oxford, OX1 3PS, United Kingdom. 8 Present address: Molecular laboratory, AZ St. Lucas Hospital, Gent, Belgium.

9 Present address: Friedrich Miescher Institute for Biomedical Research, Maulbeerstrasse 66, Basel, CH-4058, Switzerland. 10 Present address: Department of Medical Biology, International School of Medicine, ‐stanbul Medipol University, Istanbul, Turkey.

ABSTRACT

Regeneration-capable flatworms are informative research models to study the mechanisms of stem cell regulation, regeneration and tissue patterning. However, the lack of transgenesis methods significantly hampers their wider use. Here we report development of a transgenesis method for Macrostomum lignano, a basal flatworm with excellent regeneration capacity. We

demonstrate that microinjection of DNA constructs into fertilized one-cell stage eggs, followed by a low dose of irradiation, frequently results in random integration of the transgene in the genome and its stable transmission through the germline. To facilitate selection of promoter regions for transgenic reporters, we assembled and annotated the M. lignano genome, including

genome-wide mapping of transcription start regions, and show its utility by generating multiple stable transgenic lines expressing fluorescent proteins under several tissue-specific promoters. The reported transgenesis method and annotated genome sequence will permit sophisticated genetic studies on stem cells and regeneration using M. lignano as a model

organism.

INTRODUCTION

Animals that can regenerate missing body parts hold clues to advancing regenerative medicine and are attracting increased attention [1]. Significant biological insights on stem cell biology and body patterning were obtained using free-living regeneration-capable flatworms (Platyhelminthes) as models [2–4]. The most often studied representatives are the planarian species Schmidtea mediterranea [2] and Dugesia japonica [5]. Many important molecular biology

techniques and resources are established in planarians, including fluorescence-activated cell sorting, gene knockdown by RNA interference, in situ hybridization, and genome and

transcriptome assemblies [4]. One essential technique still lacking in planarians, however, is transgenesis, which is required for in-depth studies involving e.g. gene overexpression, dissection of gene regulatory elements, real-time imaging and lineage tracing. The reproductive properties of planarians, including asexual reproduction by fission and hard non-transparent cocoons containing multiple eggs in sexual strains, make development of transgenesis technically challenging in these animals.

More recently, a basal flatworm Macrostomum lignano (Macrostomorpha) emerged as a

model organism that is complementary to planarians [6–9]. The reproduction of M. lignano,

a free-living marine flatworm, differs from planarians, as it reproduces by laying individual fertilized one-cell stage eggs. One animal lays approximately one egg per day when kept in standard laboratory conditions at 20ºC. The eggs are around 100 microns in diameter, and follow the archoophoran mode of development, having yolk-rich oocytes instead of supplying the yolk to a small oocyte via yolk cells [10]. The laid eggs have relatively hard shells and can easily be separated from each other with the use of a fine plastic picker. These features make

M. lignano eggs easily amenable to various manipulations, including microinjection [11].

transparency, small size, and a short generation time of three weeks [6, 7]. It can regenerate all tissues posterior to the pharynx, and the rostrum [12]. This regeneration ability is driven by stem cells, which in flatworms are called neoblasts [3, 4, 13]. Recent research in planarians has shown that the neoblast population is heterogeneous and consists of progenitors and stem cells [14, 15]. The true pluripotent stem cell population is, however, not identified yet.

Here we present a method for transgenesis in M. lignano using microinjection of DNA into

single-cell stage embryos and demonstrate its robustness by generating multiple transgenic tissue-specific reporter lines. We also present a significantly improved genome assembly of the M. lignano DV1 line and an accompanying transcriptome assembly and genome annotation.

The developed transgenesis method, combined with the generated genomic resources, will enable new research avenues on stem cells and regeneration using M. lignano as a model

organism, including in-depth studies of gene overexpression, dissection of gene regulatory elements, real-time imaging and lineage tracing.

RESULTS

Microinjection and random integration of transgenes

M. lignano is an obligatorily non-self-fertilizing simultaneous hermaphrodite (Fig. 1a) that

produces substantial amounts of eggs (Fig. 1b,c). We reasoned that microinjection approaches used in other model organisms, such as Drosophila, zebrafish and mouse, should also work in M. lignano eggs (Fig. 1d, Supplementary Movie 1). First, we tested how the egg handling and

microinjection procedure itself impacts survival of the embryos (Supplementary Table 1). Separating the eggs laid in clumps and transferring them into new dishes resulted in a 17% drop in hatching rate, and microinjection of water decreased survival by a further 10%. Thus, in our hands more than 70% of the eggs can survive the microinjection procedure (Supplementary Table 1). When we injected fluorescent Alexa 555 dye, which can be used to track the injected material, about 50% of the eggs survived (Supplementary Table 1). For this reason, we avoided tracking dyes in subsequent experiments. Next, we injected in vitro

synthesized mRNA encoding green fluorescent protein (GFP) and observed its expression in all successfully injected embryos (n > 100) within 3 hours after injection (Fig. 1e), with little to no autofluorescence detected in either embryos or adult animals (Supplementary Fig. 1). The microinjection technique can thus be used to deliver biologically relevant materials into single-cell stage eggs with a manageable impact on the survival of the embryos.

To investigate whether exogenous DNA constructs can be introduced and expressed

in M. lignano, we cloned a 1.3 kb promoter region of the translation elongation factor 1

alpha (EFA) gene and made a transcriptional GFP fusion in the Minos transposon system

(Supplementary Fig. 2a). Microinjection of the Minos::pEFA::eGFP plasmid with or without Minos transposase mRNA resulted in detectable expression of GFP in 5-10% of the injected

lost as the animals grew (Supplementary Fig. 2f), and only a few individuals transmitted the transgene to the next generation. From these experiments we established the HUB1 transgenic line with ubiquitous GFP expression (Supplementary Fig. 2e), for which stable transgene transmission has been observed for over 50 generations[16, 17].

i

c

a

d

b

e

Figure 1 | Macrostomum lignano embryos are amenable to microinjection. (a) Schematic morphology and a bright-field image of an adult M. lignano animal. (b) Clump of fertilized eggs. (c) DIC image of a one-cell stage embryo. (d) Microinjection into a one-cell stage embryo. (e) Expression of GFP in the early embryo 3 hours after injection with in vitro synthesized GFP mRNA. Scale bars are 100 µm.

The expected result for transposon-mediated transgenesis is genomic integration of the fragment flanked by transposon inverted terminal repeats. However, plasmid sequences outside the terminal repeats, including the ampicillin resistance gene, were detected in the HUB1 line, suggesting that the integration was not mediated by Minos transposase.

Furthermore, Southern blot analysis revealed that HUB1 contains multiple transgene copies (Supplementary Fig. 2g). We next tried a different transgenesis strategy using meganuclease I-SceI[18] to improve transgenesis efficiency (Supplementary Fig. 2b). We observed a similar 3-10% frequency of initial transgene expression, and only two instances of germline transmission, one of which resulted from the negative control experiment without co-injected meganuclease protein (Supplementary Fig. 2c). These results suggest that I-SceI meganuclease does not increase efficiency of transgenesis in M. lignano, but instead that exogenous DNA can

be integrated in the genome by non-homologous recombination using the endogenous DNA repair machinery.

Table 1 | Efficiency of transgenesis with different reporter constructs and treatments Reporter Injected line Injected DNA Irradiation treatment Injected eggs Positive hatchlings (%) Germline transmission (%) Established lines EFA::eGFP DV1 PCR - 269 39 (14.50) 5 (1.86) NL1 EFA::oGFP DV1 plasmid - 114 28 (24.56) 0 -EFA::oGFP DV1 plasmid 2.5 Gy 42 13 (30.95) 2 (4.76) -EFA::oGFP DV1 fragment 2.5 Gy 102 4 (3.92) 2 (1.96) NL7 EFA::oCherry DV1 plasmid 2.5 Gy 80 4 (5.00) 1 (1.25) NL3 EFA::oCherry DV1 fragment 2.5 Gy 36 6 (16.67) 3 (8.33) NL4, NL5, NL6 EFA::H2B::oGFP DV1 fragment 2.5 Gy 38 10 (26.32) 2 (5.26) NL20 ELAV4::oGFP DV1 fragment 2.5 Gy 56 29 (51.79) 2 (3.57) NL21 MYH6::oGFP DV1 fragment 2.5 Gy 103 13 (12.62) 1 (0.97) NL9 APOB::oGFP DV1 fragment 2.5 Gy 65 2 (3.08) 1 (1.54) NL22 CABP7::oGFP DV1 plasmid - 20 2 (10.00) 1 (5.00) NL23 CABP7::oNeon Green; ELAV4:: oScarlet-I NL10 plasmid - 137 3 (2.19) 2 (1.46) NL24

Improvement of integration efficiency

The frequency of germline transgene transmission in the initial experiments was less than 0.5% of the injected eggs, while transient transgene expression was observed in up to 10% of the cases (Supplementary Fig. 2c,f). We hypothesized that mosaic integration or mechanisms similar to extrachromosomal array formation in C. elegans [19] might be at play in cases of

transient gene expression in M. lignano. We next tested two approaches used in C. elegans to

increase the efficiency of transgenesis: removal of vector backbone and injection of linear DNA fragments [20], and transgene integration by irradiation [19]. Injection of PCR-amplified vector-free transgenes resulted in the germline transmission in 5 cases out of 269 injected eggs, or 1.86% (Table 1), and the stable transgenic line NL1 was obtained during these experiments (Fig. 2a). In this line, the GFP coding sequence was optimized for M. lignano

codon usage. While we did not observe obvious differences in expression levels between codon-optimized and non-codon-optimized GFP sequences, we decided to use codon-codon-optimized versions in all subsequent experiments.

M. lignano is remarkably resistant to ionizing radiation, and a dose as high as 210 Gy is

required to eliminate all stem cells in an adult animal [8, 21]. We reasoned that irradiation of embryos immediately after transgene injection might stimulate non-homologous recombination and increase integration rates. Irradiation dose titration revealed that