Investigation of Hox Gene Expression and Wnt-Signalling in Basally Branching Ecdysozoans

Home , Antennapedia, Homeotic gene, Hox gene, Nemertodermatida, Tactopoda

Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology 1683

Investigation of Hox gene expression and Wnt-signalling in basally branching ecdysozoans

MATTIAS HOGVALL

ACTA UNIVERSITATIS UPSALIENSIS ISSN 1651-6214 ISBN 978-91-513-0363-5 UPPSALA urn:nbn:se:uu:diva-352683 2018 Dissertation presented at Uppsala University to be publicly examined in Hembergsalen, Geocentrum, Villavägen 16, Uppsala, Friday, 7 September 2018 at 10:00 for the degree of Doctor of Philosophy. The examination will be conducted in English. Faculty examiner: Dr. Andrew Peel (University of Leeds).

Abstract Hogvall, M. 2018. Investigation of Hox gene expression and Wnt-signalling in basally branching ecdysozoans. Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology 1683. 48 pp. Uppsala: Acta Universitatis Upsaliensis. ISBN 978-91-513-0363-5.

One of the most important processes in the development of an animal is the determination and patterning of the primary body axis, the anterior-posterior (AP) axis. After the AP axis has been established the embryo grows and elongates through posterior elongation. Several evolutionary conserved sets of genes and signalling pathways are involved in AP axis formation and posterior elongation, including Wnt-signalling. Wnt-signalling was involved in AP axis determination and posterior elongation even before the evolution of the Bilateria. In segmented animals, Wnt-signalling is also involved in maintaining segmental boundaries and in giving each segment its polarity. Hox genes, conversely, play a significant role in the regionalisation of the AP axis in Bilateria. This role as regionalisation factors probably emerged within the bilaterian in stem-group and it has been speculated that Wnt genes may have had this function prior to the rise of the Hox genes. The goal of this work is to shed light on the expression and function of Wnt-signalling and Hox gene patterning in basally branching ecdysozoans, Priapulida and Onychophora, two phyla that are underrepresented in current research, but represent key phyla for the understanding of ecdysozoan evolution. Wnt genes are likely to have retained a prominent function in posterior regionalisation and elongation in Priapulida. Investigation of Hox gene expression patterns proved to be difficult in Priapulida, but preliminary results suggest partially conserved function in AP axis patterning. In Onychophora, Wnt-signalling appears to be involved in segment formation, intrasegmental patterning and segment/parasegment border maintenance. Some of the onychophoran Wnt genes are expressed in Hox-like patterns suggesting a role in AP-axis patterning, a function that Wnt genes may thus have retained throughout their evolution. Finally, I have also investigated some of the factors involved in Wnt-signalling (or morphogen processing in general). These genes, the morphogen-interfering factors (MIFs), have been poorly investigated in general. I studied their expression in an onychophoran and a number of other emerging arthropod model organisms in order to obtain a more solid basis for comparison. These data, although difficult to interpret, suggest that the interaction of Wnts and MIFs is diverse and complex among Panarthropoda.

Keywords: Wnt signalling pathway, Hox genes, Fz receptors, Onychophora, Priapulida, Ecdysozoa

Mattias Hogvall, Department of Earth Sciences, Palaeobiology, Villav. 16, Uppsala University, SE-75236 Uppsala, Sweden.

ISSN 1651-6214 ISBN 978-91-513-0363-5 urn:nbn:se:uu:diva-352683 (http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-352683) List of Papers

This thesis is based on the following papers, which are referred to in the text by their Roman numerals.

I Hogvall, M., Schönauer, A., Budd, G. E., McGregor, A. P., Posnien, N. and Janssen, R. (2014) Analysis of the Wnt gene repertoire in an onychophoran provides new insights into evolution of segmentation. EvoDevo 5:14.

II Janssen, R., Schönauer, A., Weber, M., Turetzek, N., Hogvall, M., Goss, G. E., Patel, N., McGregor A. P. and Hilbrant M. (2015a) The evolution and expression of panarthropod frizzled receptors. Front. Ecol. Evol. 3:96

III Hogvall, M., Budd, G. E. and Janssen, R. (2018) Gene expression analysis of potential Wnt-signalling and Hh-signalling modifying factors in Panarthropoda. Submitted to EvoDevo.

IV Hogvall, M., Budd, G. E., Martín-Durán J. M., Hejnol, A. and Janssen, R. (2018) Expression of Priapulus caudatus Hox, Wnt, and Frizzled genes suggests partially conserved function in anterior- posterior body axis patterning and posterior elongation. Manuscript.

Reprints were made with permission from the respective publishers.

Contents

Introduction ...... 7 Some key-factors that are involved in the development and evolution of the primary body axis ...... 7 Hox genes and the primary body axis ...... 9 The multiple facets of Wnt-signalling: AP axis regionalization, posterior elongation, and boundary formation ...... 10 The main research organisms ...... 13 Priapulids ...... 13 Onychophorans ...... 17 My work at a glance ...... 19 Methods and additional data ...... 24 Animal collection, fertilization, and embryo fixation...... 24 Identification of genes ...... 25 Analysis of gene orthology ...... 25 In-situ hybridization ...... 25 Investigated genes ...... 28 Imaging ...... 29 Conclusions and future directions ...... 30 Svensk sammanfattning ...... 31 Acknowledgments...... 34 References ...... 35

Introduction

Some key-factors that are involved in the development and evolution of the primary body axis The regionalisation and pattering of the primary body axis of animals are fundamental processes for the eventual development of the embryo into its adult form. The first important point in animal development is the determination of the three body axes: which end will become the anterior and which end will become the posterior; where is dorsal and where is ventral; left side versus right side. The main body axis is often referred to as the anterior- posterior (AP) axis. After determination of this axis, additional modifications take place. The most important ones are the (posterior) elongation of the developing embryo, and the regionalisation of the body along the AP axis: in ‘segmented’ animals this is the determination of each segment’s fate. Two evolutionary conserved gene families that are involved in segmentation and posterior elongation are 1) the Hox genes, and 2) the Wnt genes (and their signalling cascades). In my thesis, I focused on the analysis of Hox gene patterns in the priapulid Priapulus caudatus and on Wnt- signalling in Panarthropoda and Priapulida. The search for the mechanisms that are responsible for the patterning of the primary body axis is one of the important questions in developmental and evolutionary research. In the last few decades, some important discoveries have been made, for example, the discovery of the conserved roles that the Hox genes play in pattering the primary axis in most of the bilaterians (e.g. Lewis 1978, Slack et al. 1993). However, other mechanisms apart from the Hox gene system also contribute to regionalisation in various types of organisms. The vinegar fly Drosophila melanogaster serves as one of the main ecdysozoan research organisms and most knowledge about developmental mechanisms comes from this species. However, Drosophila represents a long- germ band insect. This means that all segments (i.e. the entire AP body axis) are patterned and form simultaneously (e.g. Peel et al. 2005). In most other arthropods, however, posterior segments are added one by one or in pairs from a posterior located segment addition zone (SAZ).

In Drosophila, the segmentation process is under control of a hierarchical regulatory network, the so-called “segmentation gene cascade” (Nüsslein- Volhard and Wieschaus 1980, Nüsslein-Volhard et al. 1987, St Johnston and Nüsslein-Volhard 1992). The maternally inherited factor genes generate AP gradients within the egg. High concentrations of Bicoid and Hunchback gene products demarcate the anterior pole, and high concentration of the Nanos and Caudal gene products mark the posterior pole. The maternally inherited factor genes then initiate expression downward in the cascade with the gap genes, the pair-rule genes and the segment-polarity genes such as wingless/Wnt1 (wg/Wnt1), hedgehog (hh) and engrailed (en) (Rivera-Pomar and Jäckle 1996, Andrioli 2012). Last but not least, the Hox genes define most segments’ identity (reviewed in e.g. Hughes and Kaufman 2002). As mentioned above, this long-germ mode of development must be derived, since most arthropods, including most insects, generate segments from a SAZ (Davis and Patel 1999, Ten Tusscher 2013, reviewed in Liu and Kaufman 2005). This is in turn broadly similar to the way vertebrates and annelids form their segments. In arthropods, single body units are added one by one or in pairs from the SAZ, which is in the posterior region of the embryo (Davis and Patel 1999, Chipman and Akam 2008, Pueyo et al. 2008). This means that only the most anterior segments are present at the blastoderm stage of the embryo. There is a variation in how many segments that are generated at the blastoderm stage, which are categorized into short- and intermediate germ band modes. As the name implies, the intermediate germ band mode has more segments present at the blastoderm stage. These fundamental differences in the development of Drosophila compared to other arthropods (and other segmented animal groups) require partially different genetic mechanisms, especially concerning early developmental steps, i.e. the addition of new segments form the SAZ. One such pathway is represented by Delta/Notch (Dl/N)-signalling which is not involved in Drosophila segmentation, but plays a significant role in body segmentation in vertebrates (reviewed in Lai 2004, Aulehla and Herrmann 2015). It has also been shown that it plays a significant role in arthropods, as the spider Cupiennius salei, the centipede Strigamia maritima and the cockroach Periplaneta americana express the genes in the Dl/N-signalling pathway in the posterior SAZ. In the spider, there is also expression in newly formed segments (Stollewerk et al. 2003, Chipman and Akam 2008, Pueyo et al. 2008). Knock-down experiments of Dl/N-signalling in the spider have also been shown to lead to severe defects in segment pattering and formation of the segmental borders (Stollewerk et al. 2003, Schoppmeier and Damen 2005). In onychophorans, however, Dl/N-signalling does not appear to be involved in segmentation but could play a role in posterior elongation (Janssen and Budd 2016).

Because of the potential for Dl/N-signalling being involved in AP body axis development I investigated Notch (N) and Delta (Dl) in the priapulid Priapulus caudatus. Interestingly, Dl/N-signalling, Wnt-signalling, and the action of the posterior factor Caudal (Cad) are tightly intertwined in arthropods such as spiders and at least some insects that represent the short germ mode of development (McGregor et al. 2008, 2009, Fonseca et al. 2009, Chesebro et al. 2013, Schönauer et al. 2016). This highlights the complexity and suggests flexibility of gene regulatory networks (GRNs) involved in AP axis formation and patterning, although at least Cad appears to be a conserved player in posterior development more generally (e.g. Copf et al. 2004, Martin-Durán et al. 2012, Janssen and Budd 2013, El-Sherif et al. 2014).

Hox genes and the primary body axis Hox genes encode homeodomain transcription factors, and they regulate other gene expressions during development. In most animals, the Hox genes are found in a continuous cluster along the chromosome in the same order in which they are expressed along the AP axis. This is called spatial colinearity (Gaunt 1988, Akam 1989, Minguillon et al. 2005, Duboule 2007, Fröbius et al. 2008). In some animals, there is in addition a temporal colinearity, which means that the same order reflects on the timing of appearance of the genes (Dolle et al. 1989, Izpisua-Belmonte et al. 1991, Monteiro and Ferrier 2006). In segmented organisms, Hox genes determine the fate of a segment by creating a code, where different Hox genes complement each other along the anterior-posterior axis. When changes occur in the expression or activity of Hox genes, this can lead to dramatic changes in body morphology. Those can for example be homeotic transformations where extra pairs of wings develop instead of halteres (balancing organs) in the vinegar fly Drosophila melanogaster as a result of disturbed Ultrabithorax (Ubx) activity (Davis et al. 2007). Similarly, legs may form instead of antennae if the Hox gene Antennapedia (Antp) is misexpressed (Denell et al. 1981). Although the Hox genes are often found in clusters on the same chromosome, there are exceptions, for example in Drosophila, where the ancestral cluster is split into two, the Antennapedia-complex and the Bithorax-complex (Lewis 1978, Kaufman et al. 1980). The reason for the relative conservation of the clustering may lie in shared regulatory elements (Rusch and Kaufman 2000, Miller et al. 2001). The ancestral bilaterian Hox cluster contained ten Hox genes, but in some taxa, there have been losses, e.g. in Drosophila where two of the Hox genes (Hox3 and fushi tarazu) have lost their homeotic function and the expression 9

patterns have also changed (Gibson 2000). There have also been duplications of Hox clusters in some lineages and genome-wide duplications in nearly all vertebrates which gave rise to a total of 39 Hox genes divided into four separate clusters (Duboule et al. 1989, McGinnis and Krumlauf 1992, Duboule 1994, Zeltser et al. 1996). Cnidarians play a significant role in the unravelling of Hox gene evolution since data from the demosponge Amphimedon suggest that Hox genes evolved after the split from Porifera (Larroux et al. 2007). Ctenophores appear to lack Hox genes, as there is no report of any Hox gene despite several species having been sequenced (Jager et al. 2006). This lead to the belief that the emergence of the Hox-like genes probably took place in the last common ancestor of cnidarians and bilaterians. However, cnidarians do not possess all Hox classes found in protostomes and deuterostomes (=Bilateria) (Ryan et al. 2007, Chiori et al. 2009). Furthermore, Hox genes in the two branches of the Cnidaria, the anthozoans and medusozoans, appear to have a different developmental function in their lineages, as suggested by their expression patterns (Ryan et al. 2007, Chiori et al. 2009). The last common ancestor of Cnidaria+Bilateria appeared to have possessed one or two anterior and one posterior Hox gene that represent clear orthologs to the bilaterian Hox genes (Ryan et al. 2007, Chiori et al. 2009). In the acoels, which with the Nemertodermatida (Acoelomorpha) are often (but not universally) placed as a sister group to all remaining Bilateria, the Hox genes are involved in the anterior-posterior patterning of the nervous system (Hejnol and Martindale 2009). This suggests that the Hox genes may have had an ancestral function in nervous system patterning and that these genes were later co-opted for patterning other tissues and structures, including panarthropod segments. As no Hox genes are present in Porifera and Ctenophora, Wnt-patterning could be the central underlying mechanism that was responsible for the regional identity along the primary body axis, and this would suggest that the Hox genes then took over their function later in the bilaterians.

The multiple facets of Wnt-signalling: AP axis regionalization, posterior elongation, and boundary formation Wnt genes encode secreted glycoprotein ligands that bind to transmembrane receptors expressed by target cells, where they activate many interactions through a complex collection of intracellular proteins (Croce and McClay 2008). These proteins, in turn, activate or repress gene expression. In the canonical Wnt pathway, Wnt ligands bind to transmembrane receptors 10

encoded by Frizzled (Fz) genes. This leads to a release of Armadillo/β-catenin from a protein complex that otherwise promotes its degradation. The released β-catenin can bind to the transcription factor Pangolin/TCF and enter the nucleus to regulate gene expression (Croce and McClay 2008). This is not the only pathway Wnt ligands can take; they can bind to other receptors (such as Arrow and Derailed (Yoshikawa et al. 2003) or operate independently of β- catenin and TCF in non-canonical pathways (Croce and McClay 2008). Wnt-signalling, however, does not simply work via the diffusion of secreted Wnt molecules through the extracellular space and binding to their target receptors. There are multiple genetic factors that interfere with Wnt- signalling, either in a positive or a negative way. Extracellular modulators such as the secreted Frizzled-related protein (sFRP) encoding genes, or the Wnt inhibitory factor 1 (Wif-1) gene interact directly with the Wnt genes and are contributing to the fine-tuning of Wnt-signalling in at least vertebrates (Hsieh et al., 1999, Üren et al. 2000). The sFRP genes are absent from Drosophila and other insects (as far as we know) (Bastin et al. 2015), and there have not been any data on (pan)arthropod sFRP genes prior to my study (Paper III). Wif-1 interacts directly with Wnt proteins by isolating Wnt ligands and thus preventing them from binding to Frizzled (Fz) (Hsieh et al., 1999). In Drosophila, however, Shifted (Shf), which is the ortholog of vertebrate Wif-1, is implicated in Hedgehog (Hh) signalling (Glise et al. 2005, Gorfinkiel et al. 2005). The transport of Wnts is governed by the glypicans Dally and Dally-like, having a direct effect on Wnt-signalling in Drosophila (and other animals) as well (reviewed in Häcker et al. 2005, Langton et al. 2016). In any case, expression of some of these Wnt-signalling interfering factors likely play a role in AP body axis development in diverse animal groups. That was the main reason for me to study these under-investigated genes (Paper III). There are thirteen reported subfamilies of Wnt genes in metazoans (Prud'homme et al. 2002, Lengfeld et al. 2009, Cho et al. 2010). Thirteen subfamilies of Wnt genes are present in all deuterostome groups, except the loss of WntA in vertebrates (Prud'homme et al. 2002, Garriock et al. 2007, Lengfeld et al. 2009, Janssen et al. 2010a, Hogvall et al. 2014). In protostomes, twelve subfamilies have been identified for both lophotrochozoans (Cho et al. 2010) and ecdysozoans. Among ecdysozoans, there is considerable variation in the number of Wnt genes. For example, the crustacean Daphnia pulex possesses 12 Wnt genes, but the nematode worm Caenorhabditis elegans and the fly Drosophila melanogaster, only possess five and seven Wnt genes respectively (Baker 1987, Rijsewijk et al. 1987, Russell et al. 1992, Graba et al. 1995, Herman et al. 1995, Rocheleau et al. 1997, Thorpe et al. 1997, Maloof et al. 1999, Janson et al. 2001, Ganguly et al. 2005, Janssen et al. 2010a). There are also four different Fz receptors

working together with the Wnt ligands. These four Fz receptors are present in most of the hitherto investigated metazoan species, and they evolved from the two Fz genes found in sponges (Schenkelaars et al. 2015). It seems that the Fz receptors have overlapping functions in some contexts, because in knockout or knockdown studies in both Drosophila and the beetle Tribolium more than one Fz receptor had to be targeted to obtain a phenotypic change (Bhat 1998, Kennerdell and Carthew 1998, Muller et al. 1999, Beermann et al. 2011). Wnt genes appeared early in metazoan evolution, and some of their key- functions already seemed to have evolved in these early groups of animals. For example, in demosponges such as Amphimedon, expression patterns indicate a potential role of a Wnt-like gene in the patterning of the primary body axis during development, where it is expressed at the posterior end (Adamska et al. 2007). Twelve subfamilies are present in the cnidarians Nematostella vectensis and Hydra magnipapillata (Kusserow et al. 2005, Lee et al. 2006, Lengfeld et al. 2009). In N. vectensis eight of the Wnt genes are expressed along the oral-aboral axis; the expression is serial and overlapping and is similar to the expression of the Hox genes during bilaterian embryogenesis (Kusserow et al. 2005). Similarly, in the onychophoran Euperipatoides kanangrensis, several Wnt genes are expressed in a Hox gene- like pattern in young embryos (Paper I), suggesting a potential role of Wnt genes in the regionalisation of the onychophoran embryo. In cnidarians, Wnt genes are often active at the oral pole, and Wnt inhibitors such as Dickkopf are expressed aborally (Kusserow et al. 2005, Lee et al. 2006, Lengfeld et al. 2009). Wnt genes may thus be involved in the polarisation of the primary axis in the very earliest animals (prebilaterian) suggesting that there existed an ancestral posterior Wnt-signalling center that regulated many of the processes mentioned above (Petersen and Reddien 2009). In arthropods and their likely closest relatives, the onychophorans (note that the phylogenetic position of Onychophora and Tardigrada is still not fully resolved (Budd 2001, Smith and Ortega-Hernandez 2014), Wnt-signalling is involved in maintaining segmental borders and in giving each segment its polarity (Nüsslein-Volhard and Wieschaus 1980, Martinez-Arias and Lawrence 1985, Baker 1987, Martinez-Arias et al. 1988). wingless/Wnt1, the most studied Wnt gene, is present at the posterior boundary of each parasegment directly juxtaposed to cells expressing engrailed at the anterior parasegmental boundary in arthropods. This seems to be an ancestral trait of these genes in this group (Baker 1987, Nagy and Carroll 1994, Nulsen and Nagy 1999, Damen 2002, Janssen et al. 2004, 2008). The other Wnt genes are not as conserved in their expression throughout the arthropods, but most of them are either expressed as segmental stripes or in the SAZ, which could mean that they are involved in segment formation, maintenance and regionalisation of the segments (e.g. Janssen et al. 2010a).

Wnt-signalling is also involved in posterior elongation, a process that is tightly connected to AP axis development and segmentation in arthropods (e.g. McGregor et al. 2008, 2009, Bolognesi et al. 2008 Beermann et al. 2011, Williams et al. 2012, Chesebro et al. 2013, Williams and Nagy 2017). Here Wnt-signalling, Caudal and Delta/Notch-signalling interact in a gene regulatory network that controls posterior elongation (reviewed in McGregor et al. 2009).

The main research organisms Priapulids

Figure 1. The annulated body of Priapulus caudatus is divided into an anterior introvert and a posterior trunk. The introvert can retract into the trunk. In some species of priapulids such as P. caudatus a caudal appendage of unknown function is located at the posterior end of the trunk.

General information The phylum Priapulida contains only 19 described species worldwide (Shirley and Storch 1999, Todaro and Shirley 2003). They are marine worms which have been present in similar form since the Cambrian era (Conway Morris 1977, Wills 1998, Budd 2004). Priapulids are mud-dwelling or interstitial annulated worms of relatively large size (0,5 to 20 cm). They possess an anterior proboscis (or introvert) with a terminal mouth (Nielsen 2012, Schmidt-Rhaesa 2013). Most of the studies on priapulids focus on the species Priapulus caudatus Lamarck 1816 (Fig. 1) (e.g. Wennberg et al. 2008, 2009, Martín-Durán et al. 2012, 2016, Martin-Durán and Hejnol 2015, Sinigaglia et al. 2018), and report that they reproduce by external fertilisation, undergo holoblastic radial cleavage, gastrulate by invagination and epiboly, and have deuterostomic development. All of these characters are considered to be plesiomorphic features of the Ecdysozoa (Wennberg et al. 2008, 2009,

Ungerer and Scholtz 2009, Martín-Durán et al. 2012). Priapulids also show high evolutionary conservation in nuclear and mitochondrial genes (Webster et al. 2006, 2007). They resemble the predicted ecdysozoan ancestor, which is thought to have had a vermiform shape with an annulated or segmented body of relatively large adult size, radial cleavage, a terminal mouth, cycloneuralian brain, direct development and ecdysis (Budd 2001). Priapulids have both radial and bilateral symmetry (van der Land 1970, Adrianov and Malakhov 1996). The paired gonads, the ventral nerve cord and the protonephridia display bilateral symmetry, while the introvert, pharynx and the most characteristic part of the nervous system, the circum-pharyngeal “brain”, show radial symmetry (Storch 1991). In the anterior end of the body the papillae are closest to the mouth followed by scalids and pharyngeal teeth. Teeth and scalids are involved in feeding and moving, they also possess sensory functions (van der Land and Nørrevang 1985, Storch et al. 1994). The cuticle is composed of three layers; the epicuticle, the exocuticle and the endocuticle (Lemburg 1998).

Figure 2. One possible phylogenetic tree of the Ecdysozoa. Onychophorans are shown as a sister group to the arthropods, and the priapulids as part of the Scalidophora with Loricifera and Kinorhyncha as sister-groups (Modified from Giribet and Edgecombe 2017).

Phylogenetic position The Priapulida are sometimes included with the Nematoda and Nematomorpha in a group called the Cycloneuralia, where Priapulida is positioned at the base of this group (Dunn et al. 2008). Sometimes, they are placed at the bottom of the Ecdysozoa clade with Loricifera and Kinorhyncha creating the group Scalidophora (Edgecombe et al. 2011) (Fig. 2). In any case, Priapulida represents a relatively basally branching ecdysozoan group.

Development

Figure 3. Developmental stages of Priapulus caudatus (days after fertilization (DAF)). Muscular development starts five DAF with only few muscular fibres in the trunk. Six DAF the musculature forms in the introvert and the trunk, with the formation of rings and a few longitudes fibres. Seven DAF the introvert ring musculature bends upwards as the introvert starts to folds into the trunk. Eight DAF the introvert has moved inwards into the trunk completely. Nine DAF the trunk ring musculature is present in the whole embryo. Blue: trunk ring musculature. Purple: introvert ring musculature. Red: longitudinal retractor musculature (Wennberg, Budd, Hejnol, Martin-Duran, Janssen, unpublished data).

In the large macrobenthic priapulids there are many thousands of oocytes in the gonads but in the small meiobenthic priapulids only a few. The developing oocytes of P. caudatus are located in the epithelium of the gonads (Storch 1991). The cell cleavage pattern of early embryos is highly symmetrical, total, radial and stereotypical (Wennberg et al. 2008). Gastrulation occurs around three days after fertilization (DAF). The blastopore gives rise to the anus at the vegetal pole, thus following a deuterostomic pattern of development (Martín-Durán et al. 2012). Five DAF, the mouth moves upwards towards the anterior end, and the introvert grooves start to fold, creating the separation between the trunk and the introvert. A few days before hatching the introvert is totally protruded. All priapulids are believed to have larval stages. An initial mouth-less light- bulb shaped larva develops into the first loricate larva (second larval stage) (Wennberg et al. 2009, Janssen et al. 2009). This larva grows stepwise to a certain length and finally undergoes metamorphosis to form a juvenile worm.

Interestingly, final metamorphosis has never been observed, and it is thus unclear how many larval stages there are, if there is a fixed number of larval stages, and how long this development may take. The main body regions of the priapulid lorica larva are the trunk, the neck and the introvert. A rigid cuticle (i.e. the lorica) covers the trunk and, when the introvert is not protruded the introvert as well. The musculature in Priapulida consists of a system of ring muscles (Fig. 3: blue and purple) and longitudes retractor muscles (Fig. 3: red) both in the introvert and the trunk (unpublished data). The development of the muscular system starts five DAF (development at a constant temperature of 7 °C). The muscular ring system starts at six DAF to develop in both introvert and trunk. Then the longitudinal muscle retractor system develops. Eight DAF, the introvert withdraws into the trunk. At the same time the cyclopharyngeal nerve ring develops, which is seen as a ring at the anterior end with thick muscle retractor connectors. Scalids and trunk tubuli are beginning to emerge as early as six DAF. There are similarities of the priapulid muscular system to that of their closest relatives Loricifera and Kinorhyncha. Kinorhyncha have trunk longitudinal muscular structures. Also, ring muscular structures are present in the anterior region of the mouth cone, introvert and the neck region (Lang 1953, Kristensen and Higgins 1991, Müller and Schmidt-Rhaesa 2003). There are no muscular ring structures in the trunk, but there are both diagonal and dorsoventral muscle systems (Müller and Schmidt-Rhaesa 2003). Loriciferans also possess a longitudinal muscular system; there are more similarities to priapulids with muscular ring structures in both introvert and for some species even in the trunk (Kristensen 1991).

Scientific significance of Priapulida If priapulids indeed represent a slowly evolving group of ecdysozoan animals, as suggested by their morphology (Budd 2001) and sequence analysis data (Webster et al. 2006), then it is possible that they may even retain some of the molecular mechanisms underlying body patterning since their appearance early in the Cambrian (or even earlier, in the latest Ediacaran) (Huang et al. 2004). This raises the question if “ancestral” molecular patterning mechanisms could be conserved in this group of animals. Wnt-signalling, for example, appears to play an ancestral role in anterior-posterior axis formation/determination (including posterior elongation) in metazoans. It would therefore be interesting to see how Wnt genes are expressed in priapulids. Are they expressed in patterns that suggest a conserved function in posterior elongation and border formation (as for example the border between the trunk and the introvert), or may they even be expressed in Hox gene like patterns determining body regions?

Onychophorans

Figure 4. Adult female of Euperipatoides kanangrensis. The ectoderm shows no segmental features except for the segmental distribution of the limbs. The limbs are tube-like and without joints (unlike the situation in arthropods). Picture taken by Ralf Janssen.

General information The phylum Onychophora comprises around 200 described species (Oliveira Ide et al. 2012). They live in the southern hemisphere where they prefer moist areas. Most populations of onychophorans are very small (Noel Tait, personal communication). Some exceptions are represented by the Australian species living in the Blue Mountains north of Sydney. Euperipatoides kanangrensis, the onychophoran I worked on, is one such species (Fig. 4). It populates rotting eucalyptus logs where it lives in family clans that obey strict matriarchic principles. The ecology and the behavior of onychophorans is fascinating since they appear to hunt their prey similar to wolves, surrounding their prey with specific roles for certain members of the “pack”. Individuals from different packs (usually individuals from different logs) do not mix, even in captivity, and stress or the lack of food causes one pack to attack the other (Reinhard and Rowell 2005, personal observation by Ralf Janssen).

Phylogenetic position Arthropoda and Onychophora represent most likely sister phyla (Fig. 2) (Dunn et al. 2008, Campbell et al. 2011), although the position of Onychophora to Arthropoda is not fully resolved owing to the unclear position of the tardigrades (reviewed in e.g. Ortega-Hernandez et al. 2017).

Development Onychophorans comprise a wide range of developmental modes. Some species are oviparous (laying eggs), others have evolved some sort of placenta that connects the growing embryo(s) with the body of the mother, others again like Euperipatoides kanangrensis are ovoviviparous (reviewed in Anderson 17

1966, Eriksson and Tait 2012). Although they produce eggs, these develop inside the mother that then gives birth to miniature onychophorans. Early cleavages result in a blastoderm and the subsequent formation of a germ disc. A furrow forms in the center of this disc, that will later during development close in its middle and form the mouth (anterior of the groove) and the anus (posterior of the groove). Posterior to the position of the anus is the blastopore from which the divided germ band(s) of the developing embryo form (Eriksson and Tait 2012, Janssen et al. 2015b, Janssen and Budd 2017). The split germ band moves on either side of the furrow towards anterior and fuses anterior to the groove; the first and most anterior pair of somites (mesodermal blocks) harbors the frontal appendages and the eyes, the second the jaws, the third the slime papillae and the fourth to 18th the walking limbs. The mesodermal blocks and the appendages are clear features of the segmented body of onychophorans (e.g. Storch and Ruhberg 1993, Mayer et al. 2004, Mayer and Koch 2005, Mayer 2006). The ectoderm, however, does not show morphological segment boundaries as seen in arthropods (reviewed in Janssen 2017). Genetic patterning of the ectoderm via the segment-polarity genes that are involved in segment border and parasegment border formation in arthropods, however, is conserved in onychophorans (Eriksson et al. 2009, Janssen and Budd 2013). Later during development, the split germ band closes in a zipper-like manner from anterior to posterior on the ventral side. Dorsal tissue grows over the yolk and fuses at the dorsal midline, the region that also harbors the dorsal tube (heart) of the onychophoran.

Scientific significance of Onychophora Onychophorans represent highly significant research organisms, not only because they represent their own phylum, but also because they are close relatives to the arthropods, the largest and most diverse animal phylum on our planet. Understanding arthropod development and evolution very much relies on the onychophorans: first, because onychophoran data can be used to polarize data obtained from different groups of arthropods (e.g. Janssen et al. 2010b); second, many of the features seen in arthropods are assumed to represent derived features of panarthropods while onychophoran morphology may represent an ancestral state (but note that this is suggested, not proven). Such features are for example the tube-like limbs of onychophorans that may not yet have evolved joints, or the not-yet morphologically segmented ectoderm of onychophorans. If these features represent ancestral traits, as suggested by some authors, then onychophorans offer some kind of time machine allowing to investigate gene regulatory networks present in arthropod limb development and ectoderm segmentation before they evolved (Budd 1999, 2001, Jacobs and Gates 2003).

My work at a glance

Onychophoran Wnt genes (Paper I) To contribute to the vast data set of the Wnt genes of the arthropods I investigated the expression pattern of these genes in the onychophoran Euperipatoides kanangrensis. I identified 11 Wnt genes; the only Wnt that I could not find in the sequenced embryonic transcriptome is Wnt8. Several onychophoran Wnt genes show expression patterns that are similar to that of the segment polarity gene wg/Wnt1, i.e. expression in distinct transverse segmental stripes (Eriksson et al. 2009, Janssen and Budd 2013, Franke and Mayer 2014). The intrasegmental position of expression varies for these Wnt genes. Interestingly, most of the Wnt genes are present in a segment-polarity gene-like pattern only in later developmental stages, considerably later than the segment-polarity genes engrailed, wg/Wnt1 and hedgehog. This suggests that these Wnt genes are more likely involved in intrasegmental pattering instead of determining intersegmental boundaries. However, they could be involved in maintaining segmental boundaries as well. Three of the Wnt genes are expressed in broad domains with distinct anterior boundaries which are reminiscent of arthropod gap and Hox gene expression patterns. Their unique expression patterns with respect to their anterior borders of expression (compared with the expression domains of the Hox genes) could allow for the patterning of the jaw-bearing segment and the head lobes, two segments/structures that are not patterned by the Hox code.

Panarthropod Frizzled receptor encoding genes (Paper II) In order to get further insight into Wnt-signalling I also investigated the expression patterns of the Frizzled (Fz) receptors that are part of the complex system in target cells that receive Wnt ligands. The four subfamilies of Fz receptors have been characterised in a few species, which include mammals and the two arthropod model species Tribolium castaneum and Drosophila melanogaster (Bhanot et al. 1996, Kennerdell and Carthew 1998, Beermann et al. 2011). The onychophoran Euperipatoides kanangrensis possesses all four frizzled genes. Fz1 expression patterns are consistent with a role of Fz1 in segmentation across the panarthropods (Park et al. 1994, Müller et al. 1999, Beermann et al. 2011). Fz2 is not expressed in a segmental pattern which means that it is unclear if this gene played a role in segmentation in the ancestor of panarthropods. Instead, Fz2 is expressed in the median head region suggesting that this is a conserved ancestral expression domain of this gene as this is also the case for arthropods (Bhanot et al. 1996, Müller et al. 1999, Beermann et al. 2011). Euperipatoides Fz3 is expressed in transverse segmental stripes level with the position of the limb primordia, as well as in 19

the mouth and the anal valves. In Drosophila, Fz3 is expressed in segmental stripes, the eye, the leg discs and anal tissue (Sato et al. 1999, Sivasankaran et al. 2000). The ancestral role of Fz3 is still unclear, but it could have a role in the nervous system, eye and appendage development. Fz4 may be involved in nervous system development, segment formation, as well as limb development among panarthropods. I could not detect any one-to-one correlation between the expression of any given Wnt gene with any given Fz receptor. It is thus unclear what Wnt genes bind to what Fz receptor(s). In any case, I could not identify expression patterns of Fz genes resembling the Hox and gap gene like expression patterns of some of the Wnt genes (cf. Paper I).

Morphogen signalling-interfering genes (MIFs) in Panarthropoda (Paper III) I then decided to further investigate levels of Wnt-signalling. Secreted Frizzled-related protein encoding genes (sFRPs) are known to modulate Wnt- signalling in for example vertebrates (Üren et al. 2000, Bovolenta et al. 2008). Their main function is that of an antagonist binding directly to the Wnt ligands. Interestingly sFRPs are absent from insects (Bovolenta et al. 2008), and their expression has not been investigated in any other arthropod group such as myriapods and spiders. Another potential antagonist of Wnt-signalling is Wnt inhibitory factor 1 (WIF1) (Hsieh et al. 1999) which isolates Wnt ligands, preventing binding to the receptor Frizzled. I finally investigated glypicans, factors that are known to modulate Fz- dependent Wnt-signalling (Kirkpatrick et al. 2004, Han et al. 2005, Yan et al. 2009). In Drosophila, there are two glypicans, Dally and Dally-like. It is suggested that the main function of the glypicans is to regulate Wnts, Hedgehogs, fibroblast growth factors and bone morphogenetic proteins by either promoting or blocking their transport through the extracellular space and to their specific receptors. My results suggest that the investigated genes that are likely involved in morphogen interference (called morphogen interfering factors (MIFs)) are likely involved in posterior elongation, segment-border formation or/and maintenance, and limb development. All genes are expressed in very specific patterns either in transverse segmental stripes as typical for segment-polarity genes such as wg/Wnt1, or in the form of specific sectors of the limbs suggesting interaction with morphogens such as Wg, or in the posterior SAZ, suggesting a role in segment formation or/and posterior elongation. Because of the many functions these genes play in Drosophila and other animals and the scarce data on some of the genes, it is almost impossible to make clear

prediction(s) about the exact function and interaction of the MIFs with their potential target morphogens.

Hox gene, Frizzled gene, and Wnt gene expression in the priapulid Priapulus caudatus (Paper IV) A BLAST search screen for Wnt genes in sequenced embryonic transcriptomes and a preliminary sequenced genome revealed the presence of 12 Wnt genes in Priapulus. Their orthology and phylogenetic relationship to Wnt genes from other animals has been confirmed by phylogenetic analysis. Priapulus thus possesses the full set of Wnt genes reported for protostomes, as they all lack a Wnt3 gene (Janssen et al. 2010a, Cho et al. 2010). The only hitherto published expression pattern of a Wnt gene in Priapulus caudatus is that of wingless (wg/Wnt1). It is expressed around the blastopore at gastrulation and at later developmental stages around the anus. There is no expression at the border between introvert and trunk (Martín-Durán and Hejnol 2015). I obtained expression data for six more Priapulus Wnt genes, Wnt4, Wnt6, Wnt8, Wnt10, Wnt16 and WntA. All of these Wnt genes are expressed at the posterior pole of the developing embryo strongly suggesting a role in posterior development including gastrulation (as also suggested for wg/Wnt1 (Martín-Durán et al. 2012)). There is, however, variation of posterior expression, from a single expression domain at the blastopore opening (Wnt10 and Wnt16) to a more complex expression pattern with several small domains of expression around the posterior pole of the embryo (Wnt4, Wnt6 and Wnt8). RNA-seq expression data confirm that Wnt4, Wnt6, Wnt8, Wnt10, Wnt16 and WntA are expressed at higher level during embryogenesis, when compared with the other Wnt genes (Wnt5, Wnt7, Wnt11). Note that I could not obtain expression data for these weaker expressed genes. It is likely that this is because of the lower expression levels and thus technical limitations. It is surprising, however, that I could not obtain specific expression data for Wnt2 that has relatively high counts. The only maternally expressed Wnt gene is Wnt4, the other genes are not expressed before approximately three DAF. Note that Wnt9 is absent in the RNA-seq data. From the expression data obtained, I conclude that the Wnt genes likely do play a role in posterior regionalisation and posterior elongation as it is the case for other animals. Therefore, this ancestral function is likely conserved in Priapulus. However, the exact function of each Wnt gene remains unclear until further research has been undertaken. Such research could represent targeted functional studies (not available yet for any priapulid species) or general functional studies by means of chemicals (as Wnt-signalling activators like LiCl (Klein and Melton 1996) or 1-Azakenpaullone (Leost et al. 2000, Kunick et al. 2004)) that interfere with Wnt-signalling (these studies are in progress in cooperation with our colleagues at the SARS Centre in Bergen/Norway).

In Drosophila and likely also arthropods in general, and even onychophorans, wg/Wnt1 acts as a segment-polarity gene (Eriksson et al. 2009, Janssen and Budd 2013, Franke and Mayer 2014). Other Wnt genes may have a related function in determining or maintaining segmental borders (Janssen et al. 2010a, Hogvall et al. 2014). I found no evidence so far that any Wnt gene is involved in the formation of boundaries during priapulid development. One obvious such boundary is the border between the trunk and the introvert. However, none of the investigated Wnt genes are expressed in or around this border. Since it is only possible to detect gene expression at certain developmental stages, it is still possible that Wnt genes play a role in border formation, or AP axis regionalisation in other developmental stages, or that other Wnt genes of which I do not have in-situ hybridization data, have such functions. P. caudatus possesses five Frizzled genes, one in each group, with two paralogs of Fz1. The only obtained expression pattern of the Frizzled genes is that of Fz3, which is expressed in the posterior region of the embryo and reaching towards anterior along one side of the embryo; since there are no morphological landmarks, it is unclear if this is ventral, lateral or dorsal. The expression, however, appears to be restricted to the mesoderm. There are ten Hox genes in P. caudatus, however, there has been two duplication events and two losses. These duplicated Hox genes are Antp and Abd-B. The lost Hox genes are Abd-A and ftz. But note that the phylogenetic analysis of Hox genes is difficult and branch (group) support is weak. Also, Antp and ftz are generally quite similar on sequence level, and it may therefore be that one of the identified Antp genes indeed represents a derived ftz gene. Preliminary RNA-seq expression data covering a few developmental stages (as defined by days after fertilization (DAF)) suggested the possibility of temporal colinearity of P. caudatus Hox genes, as the more posterior Hox genes seem to be expressed earlier than the more posterior genes. Both lab and Hox3 are present during gastrulation (3 DAF), but lab shows much higher expression levels, later during embryogenesis Dfd becomes expressed, which is in line with potential temporal colinearity. Interestingly, pb is not expressed during embryogenesis in a high level, and this could correspond to the fact that pb does not have a function in Drosophila in early stages even though it is expressed (Randazzo et al. 1991). In order to obtain a better understanding of Hox gene mRNA distribution during priapulid development, Reverse Transcriptase PCR (RT-PCR) was conducted on total RNA isolated from every single developmental stage (unfertilized eggs, and every DAF, and including the first two larval stages) (hatching larvae and first lorica larvae). Unfortunately, the initially obtained RNA-seq data and the RT-PCR data are ambiguous showing quite different patterns of expression. In the RT-PCR experiment, the Hox genes’ expression

appear and disappear at the different developmental stages. Only data from lab and Dfd are comparable with the RNA-seq data. However, at least the absence of pb and Abd-B could be confirmed by both analyses. Reason for the discrepancies between these two analyses could be that the RT-PCR is much more sensitive and will detect even low amount of expression (or contamination). On the other hand, the RNA-seq experiment is based on quite a low number of reads (approximately 18 million reads) and because of these genes with low expression could have been missed. Despite many attempts, I only managed to visualize the expression of one single Hox gene in whole mount in-situ hybridization experiments. This gene is lab, the most anterior Hox gene. As expected, lab is present in the anterior part of the embryo in those stages where in-situ hybridization works. Interestingly, there is no expression of lab in the most anterior cells, which corresponds well with the appearance of lab in other ecdysozoans (reviewed in Hughes and Kaufman 2002).

Methods and additional data

Animal collection, fertilization, and embryo fixation Specimens of adult P. caudatus were collected from Gullmarsfjorden (Fiskebäckskil, Sweden). Gonads were dissected and kept in filtered deep seawater (fDSW). The oocytes were then released by shaking the ovaries and after cleaning several times with fDSW, active sperm from several males was used for in vitro fertilization. Fertilized eggs were kept in petri dishes filled with fDSW at a constant temperature of 9°C (7°C in the RT-PCR experiment). The eggs were washed daily with fresh fDSW to keep dishes free from contamination with bacteria, fungi and protozoans. Around 9 days after fertilization (10 days in the RT-PCR experiment), larvae hatched and these so-called hatching larvae then developed into the first lorica larvae after one week (10 days for the RT-PCR experiment) (Wennberg et al. 2011). A large number of embryos was collected of each stage (defined as day after fertilization (DAF)) for fixation. Before fixation, embryos were permeabilized with 0,05% thioglycolate, 0,01% pronase in filtered deep seawater for 45 minutes at 9°C. After several washes in fDSW, the embryos were fixed in 4% paraformaldehyde in fDSW for 1 hour at room temperature, followed by several washes in phosphate-buffered saline with 0,1% Tween-20 (PBST). Samples fixed for gene expression studies were dehydrated in 50% methanol in PBST, washed once in 100% methanol, and then stored in methanol at - 20°C. Female Euperipatoides kanangrensis were collected by Ralf Janssen in the Kanangra Boyd National Park, NSW, Australia. Dissected females carry about 50-100 embryos of consecutive developmental stages. Prior to fixation, the embryos were dissected from the uteri and the two surrounding membranes, the chorion and the vitelline membrane were removed with fine forceps. The “naked” embryos were then immediately transferred to PBST containing 4% formaldehyde. Embryos were fixed for 4-6 hours and then transferred into methanol. The embryos were stored in methanol in the freezer at -20°C for several weeks before using them in whole mount in-situ experiments.

Identification of genes Reciprocal BLAST searches were performed to identify potential orthologs of genes of interest. Onychophoran and millipede genes were identified from sequenced embryonic transcriptomes (Janssen and Budd 2013, Janssen and Posnien 2014). The priapulid genes were identified from RNA-seq data and transcriptomes that I obtained from the Hejnol group at the SARS Centre (International Centre for Marine Molecular Biology) in Bergen, Norway.

Analysis of gene orthology The amino acid sequences of P. caudatus Wnt genes, Fz genes, Hox genes (the homeodomain regions) (both Paper IV), and morphogen signaling interfering factors (MIFs) (Paper III) were aligned with multiple other species using T-Coffee followed by manual editing in SeaView (Notredame et al. 2000, Gouy et al. 2010). Bayesian phylogenetic analyses were performed with MrBayes (Huelsenbeck and Ronquist 2001) using a fixed WAG amino acid substitution model (for the Wnt genes) and a fixed LG amino acid substitution model with gamma-distributed rate variation across sites (with four rate categories). An unconstrained exponential prior probability distribution on branch lengths and an exponential prior for the gamma shape parameter for among-site rate variation was applied for all analysis. The final topology was estimated using 13,000,000 cycles for the Wnt genes, 620000 for cycles for the Hox genes and 500000 cycles for the Fz genes with MCMCMC (metropolis-coupled Markov chain Monte Carlo) analysis with four chains and the chain-heating temperature set to 0.2. The Markov chain was sampled every 200 cycles. The starting trees for the chains were randomly selected. Clade support was assessed with posterior probabilities computed with MrBayes.

In-situ hybridization In-situ hybridization was conducted with two different protocols, one for Priapulus and one for the panarthropod species I worked on. Note that I describe here the last version of both protocols; during the course of my work I modified/improved both protocols on several occasions. The protocols are similar, but the priapulid protocol is generally with longer washing and incubation steps. Priapulid embryos also require digestion with Proteinase-K for better penetration of the of the probes and antibodies. Some of the improvements to earlier used protocols are 1) adding 5% Dextran Sulfate Salt to the hybridization buffers, 2) switching from a ready to use blocking buffer to a self-mixed one that reduced background staining, 3) incubation of the

fixed embryos in H2O2 for 20 minutes in methanol prior to rehydration; this treatment increased signal intensity and reduced staining time (and thereby background signal).

Onychophorans and Priapulids arthropods*

Pre-treatment, rehydration and post-fixation

• 2% H2O2 treatment for 20 min • Stepwise rehydration from methanol • Stepwise rehydration from to PBST methanol to PBST • Digest embryos with Proteinase-K** • Fixation in 5% formaldehyde in • Stop digestion and remove charges PBST with glycine, triethanolamine and acetic anhydride • Fixation in 3,7% FA in PBST

Prehybridization • Incubate in hybridization buffer1 • Incubate in hybridization buffer2 at at room temperature room temperature • Incubate in hybridization buffer3 • Incubate in hybridization buffer2 at at 65° C 62° C overnight

Hybridization • Incubate in pre-warmed • Incubate in pre-warmed hybridization buffer3 with the hybridization buffer2 with the RNA RNA probe overnight probe for 48 hours

Probe removal • Stepwise transition from • Stepwise transition from hybridization buffer, through hybridization buffer, through saline- saline-sodium citrate solution to sodium citrate buffer to PBST PBST

Detection • Incubate in blocking buffer4 for 2 • Incubate in blocking buffer4 for 1 hours hour

• Incubate in blocking buffer4 with • Incubate in blocking buffer4 with DIG-antibody (1:2000) for 1,5 DIG-antibody (1:2000) overnight hours • Wash and incubate in PBST • Wash and incubate PBST over overnight night

Staining • Wash in staining buffer5 several • Wash in staining buffer5 several times times • Incubate in the dark in BM purple • Incubate in BM purple staining staining solution until expression solution until expression pattern pattern appears appears

* Note that this protocol was also successfully applied for embryos of the spider Parasteatoda tepidariorum, the millipede Glomeris marginata and the beetle Tribolium castaneum. ** Treatment (concentration and time) depends on batch of Proteinase-K.

Buffers/solutions

Hybridization buffer1 Hybridization buffer2 50% formamide 50% formamide 25% 20x SSC, pH 7.0 25% 20x SSC, pH 7.0 0.1% Tween-20 0.1% Tween-20 In DEPC-treated dH2O 0.05 mg/ml heparin adjust pH to 6.5 5% Dextran Sulfate Salt 0.01 mg/ml yeast RNA Blocking buffer4 0.4 mg/ml sonicated salmon sperm 1 x phosphate-buffered saline (PBS) DNA 0.02% Tween-20 In DEPC-treated dH2O 10mg/ml BSA 2% Sheep serum Hybridization buffer3 50% formamide Staining buffer5 20% 20x SSC, pH 6.5 100 mM Tris pH 9.5 0.1% Tween-20 150 mM NaCl 0.05 mg/ml heparin 10 mM MgCl2 5% Dextran Sulfate Salt 0.1% Tween-20 1% SDS In DEPC-treated dH2O 100 µg/ml sonicated salmon sperm DNA In DEPC-treated dH2O 27

Investigated genes

Genes Species Cloned Expression pattern Wnt2 Ek1, Pc2 Yes1 Yes1 Wnt4 Ek1, Pc2 Yes1-2 Yes1-2 Wnt5 Ek1, Pc2 Yes1 Yes1 Wnt6 Ek1, Pc2 Yes1-2 Yes1-2 Wnt7 Ek1, Pc2 Yes1-2 Yes1 Wnt8 Pc Yes Yes Wnt9 Ek1, Pc2 Yes1-2 Yes1 Wnt10 Ek1, Pc2 Yes1-2 Yes1-2 Wnt11 Ek1, Pc2 Yes1-2 Yes1 Wnt16 Ek1, Pc2 Yes1-2 Yes1-2 WntA Ek1, Pc2 Yes1-2 Yes1-2 Fz1.1 Ek1, Pc2, Pt3, Gm4 Yes1-4 Yes1, 3-4 Fz1.2 Pc - - Fz4.1 Ek1, Pc2, Pt3, Gm4 Yes1, 3-4 Yes1, 3-4 Fz4.2 Pt Yes Yes Fz2 Ek1, Pc2, Pt3, Gm4 Yes Yes1, 3-4 Fz3 Ek1, Pc2, Gm4 Yes1-2, 4 Yes1-2, 4 lab Pc Yes Yes pb Pc - - Scr Pc Yes - Antp1 Pc Yes - Antp2 Pc Yes - Hox3 Pc Yes - Dfd Pc Yes - Ubx Pc - - Abd-B2 Pc Yes - Abd-B1 Pc Yes - en Pc Yes - hh Pc Yes - Notch Pc Yes - Delta Pc Yes - dally Ek1, Gm4, Pt3, Tc5 Yes1, 3-5 Yes1, 3-5 dlp1 Ek1, Gm4, Pt3, Tc5 Yes1, 3-5 Yes1, 3-5 dlp2 Pt Yes Yes sFRP125 Ek1, Gm4, Pt3 Yes1, 3-4 Yes1, 3-4 sFRP34 Ek1, Gm4 Yes1, 4 Yes1 shf Ek1, Gm4, Pt3, Tc5 Yes1, 3-5 Yes1, 3-5

Imaging Embryos of Priapulus caudatus were cleared in 70% glycerol in PBST and imaged with a Leica Leitz DMRXE microscope with a Leica DFC550 camera using bright field Nomarski optics. Embryos of Parasteatoda tepidariorum, Glomeris marginata, Tribolium castaneum and Euperipatoides kanangrensis were photographed using a Leica DC100 digital camera mounted onto a Leica dissection microscope. Embryos of Tribolium castaneum were taken up in 87% glycerol and yolk was removed. These embryos were then mounted on glass slides under a thin glass cover. Prior to the dissection of limbs of Parasteatoda, embryos were taken up in 87% glycerol. Pictures were modified in Fiji and Photoshop CS6 (Adobe), and figures were made using either Photoshop CS6 or Illustrator CS6 (Adobe).

Conclusions and future directions

I have shown that most of the Wnt genes are expressed in segmental patterns in the onychophoran, suggesting potential roles in intrasegmental patterning and the maintenance of segmental and/or parasegmental boundaries. Additionally, some of the Wnt genes are expressed in the posterior segment addition zone, suggesting a potential function in posterior elongation. Finally, a few Wnt genes also exhibit Hox gene-like patterns, suggesting a potential function in AP body regionalisation and segment identity. This role of the Wnt genes could be seen as ancestral that is not present in any hitherto investigated arthropod species. Further investigation of Wnt-signalling by means of expanding my research to potential Wnt interfering factors (aka morphogen interfering factors MIFs) in the onychophoran and several arthropods revealed a likely function of these genes in segmentation and posterior elongation. However, patterns reminiscent of those Hox-like patterns of some Wnt genes in the onychophoran were not found. In the more basally branching priapulid, the Wnt gene are exclusively expressed (in the investigated stages) in posterior tissue surrounding the blastopore, suggesting a function in posterior elongation. Such posterior function of Wnt genes (or Wnt-signalling in general) appears to represent an ancestral function. My preliminary data on priapulid Hox genes are interesting as they suggest a limited input of Hox genes in AP body axis regionalization. However, this part of my research will require further work. For the future, it would be important to fill the gaps about our knowledge of Wnt and Hox gene expression (and potentially function) in the priapulid. However, for technical reasons this could be difficult. It would also be useful to further investigate the expression patterns of MIFs by means of double in- situs in the arthropods and the onychophoran. For the beetle and the spider, functional studies could be performed to obtain insight into the function of these genes; this is currently not possible for any priapulid, myriapod or onychophoran.

Svensk sammanfattning

Hur djur får sin kroppsform har länge varit ett hett forskningsämne. En av de stora upptäckterna inom detta område var när de så kallade Hox-generna hittades; det visade sig att denna familj av gener bestämde vad i kroppen som skulle utvecklas till vad. Med andra ord hade dessa gener ansvar för i vilken riktning olika delar av kroppen skulle utvecklas, till exempel vilka celler som skulle bli ben eller vilka celler skulle bli ögon. Det har också visat sig att dessa geners position på kromosomerna har en påverkan på hur generna sedan uttrycks – detta fenomen påverkar t.ex. i vilken ordning de uttrycks på kroppen (även kallad rumslig kollinjäritet) och när de ska uttryckas (även kallad timlig kollinjäritet). En annan stor upptäckt angående Hox-generna är att de existerar och har liknande egenskaper i nästan alla tvåsidiga djur (Bilateria (invertebrater och vertebrater)). Däremot saknades dessa Hox-gener i de djur som existerade innan de tvåsidiga djurens uppkomst, vilket tyder på att dessa gener måste ha uppstått i den evolutionära brytningen mellan de tvåsidiga djuren och dess föregångare. I dessa föregångare återfanns däremot spår av andra gener som skulle kunna ha varit Hox-genernas förfäder, vilka dock saknade Hox-genernas egenskaper. Detta skvallrade om en annan gengrupp med likande egenskaper som Hox-generna, med egenskapen att regionalisera djurens kropp, innan Hox-generna fick denna uppgift. En gengrupp som i leddjur (Arthropoda) har med segmentering att göra är Wnt- generna där den mest studerade genen i denna grupp heter wingless. Denna gen är delansvarig i att uppehålla de molekylära segmentgränserna hos segmenterade djur (till exempel leddjur och ringmaskar). Wnt-generna uppkom väldigt tidigt i evolutionen och kan hittas i väldigt primitiva djur. I dessa primitiva djur verkar dock Wnt-generna ha en annan egenskap än i segmenterade djur där de primärt har en koppling till den bakre delen av djuret inom utvecklingen av dessa. Det har föreslagits att dessa Wnt-gener hade med regionaliserande att göra inom dessa primitiva djur, en egenskap som sedan togs över av Hox-generna. Men hur denna övergång såg ut och ifall dessa Wnt-gener har kvar lite av denna egenskap i mer utvecklade grupper av djur är fortfarande ett frågetecken. För att undersöka vad som styrde regionaliseringen, och senare styrningen av mer komplexa djurformer, har vi använt oss av både penismaskar (Priapulida) och en art av sammetsmaskar (Onychophora). Penismaskarna 31

lever marint i sedimenten och finns över hela jordklotet. De kan vara allt mellan någon millimeter upp till emot 30 cm långa och består av en bål och ett introvert som kan fällas in i bålen och det är med hjälp av denna introvert som de förflyttar sig samt angriper föda. Penismaskarna är en väldigt primitiv art som existerat länge och är därför intressant att undersöka ur ett evolutionärt perspektiv. Dessvärre finns det inte mycket information om dessa primitiva maskar. Den andra gruppen modelldjur som valts att undersökas, sammetsmaskarna, lever i tropiska miljöer på land och finns i den södra hemisfären. De är förmodligen en systergrupp till leddjuren och är som leddjuren segmenterade. Dock har de inget hårt ytterhölje som leddjuren men det tydligaste beviset att de är segmenterade är de ben som finns på varje segment. Det faktum att det finns så lite information kring penismasken har gjort att de metoder (in-situ hybridisering) som använts inte varit helt optimerade för denna djurgrupp. Trots dessa svårigheter har vi visat att Wnt generna har polariserande egenskaper i dessa genetiskt konserverade djur. Sex av de totalt 12 existerande Wnt generna i penismask visade sig uttryckas i bakre delen av kroppen. Den variation som observerats i uttrycken kan ses som ett bevis på att de har olika egenskaper i att polarisera olika delar av bakkroppen. För Hox generna så har vi bara fått tag på ett utryck och det är den genen som är längst fram (labial). Detta uttryck är jämförbart med de utryck som återfinns i tex leddjur, vilket antyder att de har liknande egenskaper även i penismasken. Vi har även fått data från stadiebaserad RNA sekvensering som kan visa när, och hur mycket, gener utrycks. Denna data kan dock inte visa gener som utrycks i låg mängd. För Wnt generna stämmer våra data punkter relativt bra överens med in-situ hybridiseringen, då vi hittar utryck i embryona för de gener som har högt utryck i RNA sekvenseringen. För Hox generna är det dock skillnader då vi inte hittar uttryck i in-situ hybridisering för några som har ganska högt utryck i RNA sekvenseringen. Det kan dock vara andra förhållanden som på verkar våra experiment. Sammetsmaskarna utrycker Wnt generna i ett segmenterande mönster, vilket antagligen betyder att de har med segmentering av djuret att göra. Vissa av dessa gener är även utryckta i den tillväxtzon som finns i den bakre delen av sammetsmaskarnas kropp under utvecklingen. De är inte utryckta i samma del av varje segment, vilket föreslår att de har lite olika egenskaper inom varje segment. Intressant nog visade det sig att några av Wnt generna hade ett mer Hox liknande uttrycksmönster; genuttrycken uppvisade starka främre gränser i unga sammetsmaskembryon. Detta kan vara bevis på att Wnt generna har en regionaliserade egenskaper även i sammetsmaskar – en egenskap som dessa gener sedan förlorat hos leddjuren. Vi har även studerat Wnt genernas receptorer (Frizzled) och har inte hittat något klart och tydligt samband mellan

specifika Wnt gener och de existerande fyra Frizzled-receptorer, vilket skulle kunna tolkas som att Wnt generna kan intrigera med alla dessa fyra receptorer. Dessa signalsystem behövs vanligtvis finjusteras och begränsas vid de delar av kroppen som inte ska innehålla dessa. Här kommer modulatorer in, som kan transportera eller begränsa Wnt proteinernas uttryck. Vi valde att studera fem modulatorer och dess inverkan på signalsystemen. Det har dock visat sig att de har en väldigt anpassningsbar funktion vilket tyder på att Wnt signalsystemet är otroligt komplext och att mer forskning på detta område är högst önskvärt.

Acknowledgments

I would like to thank my supervisors, Graham Budd, Andreas Hejnol and Ralf Janssen. Especially Ralf always encouraged me when nothing was working in the lab and helped me whenever I needed. I would like to thank the people in the Sars Centre in Bergen for their company during several periods of fieldwork on the West Coast in Sweden and for their warm welcome and help during my stay in Bergen. I would like to especially thank Chema for all the help showing me how to work with the penisworms and all the helpful mails over the years. I would like to thank all the helpful people at Sven Lovén Centre Kristineberg in Fiskebäckskil, where the collection of the penisworms took place, and all the people that helped me catching the Baltic penisworm at Askölaboratoriet (Stockholm University). I would like to thank all the people in the Palaeobiology program over the years I have been there. I would like to thank Johan Svensson and Gaëtan Philippot for the endless lunches and talks: without that this PhD would not be the same. I would like to thank all of my other friends that have joked at my expense over the fact that I have been working on penisworms. I would like to thank my family for all the support and specially my wife (Marie) and kids (Ida and Line) for their love and support during this time.

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