Analysis of Wnt ligands and Fz receptors in Ecdysozoa: investigating the evolution of segmentation. by Mattias Hogvall

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1 Analysis of Wnt ligands and Fz receptors in Ecdysozoa: investigating the evolution of segmentation. by Mattias Hogvall

2 Abstracts Paper I Hogvall, M., Schönauer, A., Budd, G. E., McGregor, A P., Posnien, N. and Janssen, R Analysis of the Wnt gene repertoire in an onychophoran provides new insights into evolution of segmentation. EvoDevo, 5:14. The Onychophora are a probable sister group to Arthropoda, one of the most intensively studied animal phyla from a developmental perspective. Pioneering work on the fruit fly Drosophila melanogaster and subsequent investigation of other arthropods has revealed important roles for Wnt genes during many developmental processes in these animals. We screened the embryonic transcriptome of the onychophoran Euperipatoides kanangrensis and found that at least 11 Wnt genes are expressed during embryogenesis. These genes represent 11 of the 13 known subfamilies of Wnt genes. Many onychophoran Wnt genes are expressed in segment polarity gene-like patterns, suggesting a general role for these ligands during segment regionalization, as has been described in arthropods. During early stages of development, Wnt2, Wnt4, and Wnt5 are expressed in broad multiple segment-wide domains that are reminiscent of arthropod gap and Hox gene expression patterns, which suggests an early instructive role for Wnt genes during E. kanangrensis segmentation. 2

3 Paper II Janssen, R., Schönauer, A., Weber, M., Turetzek, N., Hogvall, M., Goss, G E., Patel, N., McGregor A P. and Hilbrant M The evolution and expression of pan-arthropod frizzled receptors. Front. Ecol. Evol. 3:96. Wnt signalling regulates many important processes during metazoan development. It has been shown that Wnt ligands represent an ancient and diverse family of proteins that likely function in complex signalling landscapes to induce target cells via receptors including those of the Frizzled (Fz) family. The four subfamilies of Fz receptors also evolved early in metazoan evolution. To date, Fz receptors have been characterized mainly in mammals, the nematode Caenorhabditis elegans and insects such as Drosophila melanogaster. To compare these findings with other metazoans, we explored the repertoire of fz genes in three panarthropod species: Parasteatoda tepidariorum, Glomeris marginata, and Euperipatoides kanangrensis, representing the Chelicerata, Myriapoda, and Onychophora, respectively. We found that these three diverse panarthropods each have four fz genes, with representatives of all four metazoan fz subfamilies found in Glomeris and Euperipatoides, while Parasteatoda does not have a fz3 gene, but has two fz4 paralogs. Furthermore, we characterize the expression patterns of all the fz genes among these animals. Our results exemplify the evolutionary diversity of Fz receptors and reveals conserved and divergent aspects of their protein sequences and expression patterns among panarthropods; thus providing new insights into the evolution of Wnt signalling more generally.

4 List of Papers This thesis is based on the following papers, which are referred to in the text by their Roman numerals. 1. 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. (2015) The evolution and expression of panarthropod frizzled receptors. Front. Ecol. Evol. 3:96. 4

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6 Contents Abstracts... 2 List of Papers... 4 Introduction... 8 Segmentation... 8 The origin of segmentation... 9 Common origin?... 9 Independent evolution of the segmented body? Arthropod segmentation Onychophoran segmentation The role of Wnt ligands and Fz receptors in segmentation Hox genes Priapulida: a basally branching ecdysozoan group Published data Paper I Paper II Unpublished data Hitherto investigated genes Expression patterns of Wnt genes Expression patterns of Hox genes Future directions Acknowledgments References

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8 Introduction Segmentation How the body plan of an organism is organised will play an important role in how successful the organism will be when adapting to new habitats and competing with other organisms. Segmentation is a body organisation that is present in several groups of complex animals. A segmented body plan consists of distinct similar units: the segments, each containing a set of serially homologous organs. (Budd 2001, Scholtz and Edgecombe 2005) Since segments are relatively autonomous units they can evolve more freely and this could lead to faster adaptation. Segmentation has often been discussed in the contexts of developmental biology and evolution. One of the big questions in that field is if segmentation evolved independently in clearly segmented phyla like annelids, arthropods and vertebrates or if either the ancestor of the bilaterians or the protostomes was already segmented (Davis and Patel 1999). As stated previously, the arthropods, annelids and vertebrates are the phyla that are usually considered to be segmented. It is important to understand this from an evolutionary point of view, since it is very unlikely that the stem groups and sister groups to the segmented groups did not contain any segmented features (Budd 2001). Therefore, as segmentation is an evolutionary feature, there must be intermediate forms that evolved into the obviously segmented body of the annelids, arthropods and vertebrates. Or if the various morphological segmental features have been lost or reduced in these intermediate forms there may be at least leftovers of the predicted underlying genetic network. In phyla related to arthropods and annelids there are at least some possibly segmental morphological structures. The tardigrades show a segmented ectoderm (Eibye-Jacobson 1997) and the onychophorans show some segmental features, but lack grooves in the ectoderm between segments (discussed further below). The molluscs have a segmented musculature and external sclerites in the polyplacophorans and monoplacophorans, but lack a segmented coelom (Jacobs et al. 2000). 8

9 The origin of segmentation Common origin? The vertebrates, annelids and arthropods are not closely related and several unsegmented organisms are clearly closer related to them than they are to each other (Fig. 1). Therefore, the conclusion that segmentation of these groups originated from a common ancestor could be difficult to prove. It was thought earlier that annelids and arthropods were sister groups because they shared a segmented body (the group Articulata), but this idea was abandoned with the advent of modern DNA/RNA-sequence based phylogenetic analysis (Aguinaldo et al 1997, Peterson and Eernisse 2001). Nevertheless, there are some morphological similarities concerning the mechanism of segment addition and the genes controlling this process in all the segmented phyla. Figure 1. Phylogenetic tree of Bilateria. In red are the groups that have a clear segmental body pattern (Modified from Edgecombe et al ). For example, the Notch signaling pathway plays a significant role in body segmentation in vertebrates (reviewed in Aulehla and Herrmann 2015, Lai 2004). Furthermore, it has been shown that the spider Cupiennius salei, the centipede Strigamia maritima and the cockroach Periplaneta americana express the genes in the Notch signaling pathway (including the ligand Delta) in the posterior segment addition zone. In the spider there is also expression in newly formed segments (Chipman and Akam 2008, Pueyo et al. 2008, Stollewerk et al. 2003). Functional studies in the spider have revealed that knock-down of Delta/Notch signaling leads to severe defects in 9

10 segment pattering and formation of the segmental borders (Stollewerk et al. 2003, Schoppmeier and Damen 2005). This could be evidence that arthropods share some mechanism with vertebrates that could have originated from a common ancestor. Meanwhile, in Drosophila the Notch signaling pathway is not known to be required in segmentation, but has a crucial role in boundary formation in other structures (Papayannopoulos et al. 1998, de Celis et al. 1998). Annelids and arthropods also share some molecular mechanisms involved in the segmentation process. The annelid Platyneris dumerilii shows a similar expression pattern of the segment polarity genes (SPG) wingless, engrailed and hedgehog as arthropods and onychophorans (Baker 1987, Prud homme et al. 2003, Eriksson et al. 2009, Dray et al. 2010, Janssen and Budd 2013). In Drosophila, the genes engrailed and wingless are expressed at the parasegment boundaries juxtaposed to each other; the same pattern is present in onychophorans. The similarities of the expression between arthropods and onychophorans may suggest that this relationship between these genes predates the origin of arthropods. If the annelid data are included, the origin of these relationships could even go back to the common ancestor of Protostomia. However, the annelid data are still under debate since no other group of annelids shares these conserved expressed patterns (Seaver and Kaneshige 2006). So in the light of these shared mechanisms, segmentation could have evolved once. This may seem unlikely if you look at all the phyla that then would have lost this feature. Independent evolution of the segmented body? Another alternative to the view that segmentation evolved only once and was then lost on multiple occasions is that the segmented phyla evolved independently using a common molecular toolbox, which could explain the shared molecular processes. As stated above, the Notch signaling pathway has a role in other boundary formations and that could be evidence that the toolbox is easy to incorporate for these types of developmental processes. A similar example of convergent evolution is the development of the cameratype eye in vertebrates and the cubozoan jellyfish Tripedalia cystophora. The latter one uses similar developmental foundations as the vertebrates, which are the phototransduction cascade and the melanogenic pathway (Kozmik et al. 2008). 10

11 Arthropod segmentation The phylum Arthropoda includes the hexapods, chelicerates, myriapods and crustaceans (Fig. 2). An arthropod is an invertebrate animal with an exoskeleton made of chitin, a segmented body, and jointed appendages. They are also the most successful phylum in terms of species number and number of individuals; they also have a rich fossil record. On top of this, arthropods have managed to spread to all different habitats around the world. One thought on this is that their segmented body may have led to this evolutionary success. Figure 2. Phylogenetic tree of Arthropoda The fruit fly (Drosophila melanogaster), which is a long germ band insect, generates its segments almost simultaneously during embryogenesis from a syncytial blastoderm. This mode of development is not only present in Drosophila, but also in other insects such as the beetle Bruchidius obtectus and the honeybee Apis mellifera (reviewed in Davis and Patel 1999). It seems that is has evolved independently several times connected to the need for rapid development (reviewed in Ten Tusscher 2013). In Drosophila, the segmentation process is under control of a hierarchical regulatory cascade, starting with the maternally inherited factor genes, which generate gradients within the egg. High concentrations of the Bicoid and Hunchback gene products demarcate the anterior pole, and high concentration of the Nanos and Caudal gene products mark the posterior pole of the embryo. The maternally inherited factor genes then initiate expression downward in the cascade with the gap genes, which divides the embryo into broad multi-segment wide domains along the anterior-posterior axis. Subsequently the pair-rule genes subdivide the gap gene domains into smaller segmental stripes and define the parasegmental boundaries. The last 11

12 molecular level in the tier is that of the segment polarity genes. These genes act to maintain the segmental boundaries and define the polarity of the segments (Rivera-Pomar and Jäckle 1996, Andrioli 2012). This long-germ mode of development, however, is derived since most arthropods, including most insects, generate segments from a posterior segment addition zone (Ten Tusscher 2013, Davis and Patel 1999, reviewed in Liu and Kaufman 2005). And this is indeed similar to the way vertebrates and annelids form their segments. Single body units are added one by one or in pairs from the segment addition zone, 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 shortand intermediate germ band modes. As the name implies the intermediate germ band mode has more segments present at the blastoderm stage. Despite the different developmental modes, many of the genes that are involved in Drosophila segmentation are conserved in both systems (Ten Tusscher 2013), especially concerning the function of the segment polarity genes and pair-rule genes (reviewed in Liu and Kaufman 2005). When it comes to the maternally inherited factor genes and the gap genes there are fewer similarities, for example the bicoid gene is absent outside cyclorrhaphan flies (which includes Drosophila) (Stauber et al. 2000). Onychophoran segmentation Arthropoda and Onychophora are most likely sister phyla (Dunn et al. 2008, Zantke et al. 2008) (Fig. 3), although the possibility that tardigrades are sister group to arthropods cannot be discounted (Budd 2001, Smith and Ortega-Hernandez 2014). Therefore, the onychophorans represent a suitable model group when investigating the ancestral arthropod segmentation mechanisms and their origin. Onychophorans are terrestrial and live in the tropical part of the southern hemisphere and consist of around 200 described species (Olivera Ide et al. 2012). 12

13 Figure 3. Phylogenetic tree of the Ecdysozoa. Showing the onychophorans as a sister group to the arthropods (Modified from Edgecombe et al ). Onychophorans are segmented, but have soft bodies, and could therefore resemble an intermediate between the arthropods and their likely worm-like ancestors (Fig. 4). They display characteristics that may be close to the form of the last common ancestor of onychophorans and arthropods. However, onychophorans have had as much time to diverge as arthropods, and the radiation of the arthropods does not necessarily mean that their body plan is more derived. In onychophorans the trunk mesoderm is overtly segmented and forms clear somites. On a molecular level the pattering of the segment polarity genes (SPGs) is also clearly segmental and identical to SPG patterning in arthropods (Eriksson et al. 2009, Janssen and Budd 2013). However, the ectoderm is not segmented with the position of the limbs being the only visible segmental pattern (Storch and Ruhberg 1993, Mayer et al. 2004, Mayer and Koch 2005, Mayer 2006). It is still unclear whether the onychophorans' body plan is ancestral to the arthropods or a derived condition unique to the onychophorans (Budd 1999, 2001, Jacobs and Gates 2003; but see also Smith and Ortega-Hernandez 2014 suggesting it is partly derived). 13

14 Figure 4. Euperipatoides kanangrensis. The ectoderm shows no segmental features except the repeated limbs. Picture taken by Ralf Janssen. The segments of onychophorans are added from the segment addition zone in the posterior part of the body, similar to most segmented organisms. As said above, the expression of wingless and engrailed in the onychophoran species Euperipatoides kanangrensis is very similar to the expression in arthropods (Eriksson et al. 2009). Other segment polarity genes also seem to be conserved between the arthropods and the onychophorans (Janssen and Budd 2013). This suggests that the segment polarity gene network and its function are conserved in arthropods and onychophorans. The role of Wnt ligands and Fz receptors in segmentation 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. Wnt ligands bind to transmembrane receptors 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 enters 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 ligands not only act through different pathways, but there are also 13 different 14

15 subfamilies of Wnt genes in metazoans (Cho et el 2010, Lengfeld et al. 2009, Prud homme et al. 2002, Janssen et al. 2010). 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 was required to be knocked down to get a phenotypic change (Bhat 1998, Kennerdell and Carthew 1998, Muller et al. 1999). The Wnt gene subfamilies appear very early in evolution, and it seems that the expansion of the Wnt genes occurred after the divergence of ctenophores and sponges (which have 3-4 Wnt genes) as in Cnidaria 12 are found (Lengfeld et al. 2009, Kusserow et al. 2005, Lee et al. 2006, Pang et al. 2010). The starlet sea anemone Nematostella vectensis expresses eight genes along the oral-aboral axis, either at the oral end, or in staggered arrays along the axis (Kusserow et al. 2005). The expression is serial and overlapping along the oral-aboral axis of the embryo and is reminiscent of the Hox pattern in bilaterian embryogenesis. In Protostomia the Wnt3 gene has been lost. As a consequence, in Lophotrochozoa there are 12 Wnt genes (Cho et el. 2010), as in most of the Ecdysozoa. While this is still the case in the crustacean Daphnia pulex (Janssen et al. 2010), in the closely related insects there has been a severe loss of Wnt genes. Drosophila only has seven Wnt genes left (Baker 1987, Ganguly et al. 2005, Graba et al. 1995) and Tribolium has nine left (Bolognesi et al. 2008). It is not known if the present Wnt genes have taken over the functions of those Wnt genes that have been lost. Wingless, as said above, is involved in maintaining segmental borders and in giving each segment its polarity; it specifies and maintains boundaries and cell fates in the primary segmental units or parasegments (Baker 1987, Martinez-Arias and Lawrence 1985, Martinez-Arias et al. 1988, Nüsslein- Volhard and Wieschaus 1980). Wingless is the most studied Wnt gene. It is expressed at the posterior boundary of each parasegment directly juxtaposed to cells expressing engrailed at the anterior parasegmental boundary. This seems to be an ancestral trait of these genes in arthropods (Baker 1987, Nagy and Carroll 1994, Nulsen and Nagy 1999, Damen 2002, Janssen et al. 2004, Janssen et al. 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 segment addition zone, which could mean that they are involved in segment formation, maintenance and regionalization of the segments (Janssen et al. 2010). In the onychophorans several Wnt genes are expressed in Hox gene-like patterns (Hogvall et al 2014). This is similar to the above-mentioned Hox gene-like patterns in the basally branching Nematostella. This finding could indicate an ancestral function of Wnt genes in patterning/regionalizing the 15

16 anterior-posterior body axis in animals prior to the evolution of the Hox gene system. Hox genes Hox genes encode homeodomain transcription factors and act together to determine body patterns along the anterior-posterior axis of an organism (Botas and Auwers 1996). 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. In general, a more posterior Hox gene will suppress the expression or the function of a more anterior one (Miller et al. 2001). When changes occur in the expression or activity of Hox genes, it can lead to evolutionary changes in the body morphology. When mutations or knockdowns of single Hox genes occur, one body part can be replaced by another. For example, extra pairs of wings instead of halteres in Drosophila (caused by mis-expression of the Ultrabithorax gene). This is called homeotic function and is one characteristic of the Hox genes. The Hox genes are found in clusters, mostly on the same chromosome, but there are exceptions as in the flatworm Schistosoma mansoni, which has four Hox genes that are dispersed on two different chromosomes (Pierce et al. 2005). In most animals the Hox clusters are fragmented, but there are some that have retained (or formed) an intact cluster (Minguillon et al. 2005, Duboule 1994). The ancestral bilaterian Hox cluster had ten Hox genes, but in some taxa there have been losses, e.g. in Drosophila where two of them (Hox3 and fushi tarazu) have lost their homeotic function and the expression patterns have also changed (Gibson 2000). There have also been duplications of Hox clusters in some lineages. The Hox genes show both spatial and in some lineages temporal colinearity (mainly vertebrates). Spatial colinearity means that the gene order on the chromosome 5 to 3 reflects the order of gene expression along the anterior-posterior body axis (Kaufman et al. 1990). Temporal colinearity means that the most anterior genes are expressed earlier than the posterior genes (Duboule 1994, Kmita and Duboule 2003). In arthropods and onychophorans the Hox genes are very conserved (reviewed in Hughes and Kaufman 2002, Janssen et al. 2014). In basally branching ecdysozoans, the priapulids, there is indication that the species Priapulus caudatus has ten Hox genes (de Rosa et al. 1999), but Abdominal- A is missing. This may be the ancestral number of Hox genes in the ecdysozoans. 16

17 Priapulida: a basally branching ecdysozoan group The phylum Priapulida contains of 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 (ConwayMorris 1977, Wills 1998, Budd 2004). The knowledge of priapulids is mostly based on studies on Priapulus caudatus, which is mud dwelling and lives in the northern hemisphere. Priapulids also show a high evolutionary conservation in nuclear and mitochondrial genes (Webster et al. 2006, Webster et al. 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 (Fig. 5)(Budd 2001). Figure 5. Adult specimen of Priapulus caudatus. Its body is divided into an anterior introvert and a posterior trunk. The introvert can retract inside the trunk. In some species as in P. caudatus a caudal appendage is present with a probably respiratory function (Fänge and Mattison 1961). 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 it is positioned at the base of the Ecdysozoa clade with Loricifera and Kinorhyncha creating the group Scalidophora (Edgecombe et al. 2011)(fig. 3). In any case, Priapulida represents a basally branching ecdysozoan group. An adult priapulid has a worm shaped body with an anterior introvert and posterior trunk. The boundary between the introvert and the trunk could 17

18 resemble a segment boundary, but no indication for that is known yet. The only hitherto published expression pattern of a segment polarity gene in Priapulus caudatus is that of wingless (wg/wnt1). It is expressed around the blastopore at gastrulation and around the anus when the introvert has developed in older stages. There is no expression around the borders between introvert and the trunk region (Martín-Duran and Hejnol 2015). However, that does not necessarily mean that no Wnt gene, or no SPG is actually involved in the formation of this morphological boundary,. Also, if the ancestor of the priapulids was segmented, then it could be possible that remnants of the underlying gene regulatory network are still in place, possibly recruited for other/similar (segmental) functions. 18

19 Published data Paper I To contribute to the vast data of arthropod Wnt genes we investigated the expression the expression patterns of these genes in the onychophoran Euperipatoides kanangrensis. We found 11 Wnt genes, which includes all expected orthologs except for Wnt8. Several onychophoran Wnt genes are expressed in a segment polarity genelike pattern with transverse segmental stripes. They differ in position where in the segment they are expressed (Fig. 6). Interestingly, most of the Wnt genes are expressed in a segment polarity gene-like pattern only in later developmental stages, considerably later than the segment polarity genes engrailed, wingless and hedgehog. This suggests that the Wnt genes are more likely involved in intrasegmental pattering instead of determining intersegmental boundaries. However, they could be involved in maintaining segmental boundaries. Figure 6. Segment polarity gene-like intrasegmental expression of Wnt genes. Four of the Wnt genes are expressed in broad domains with distinct anterior boundaries which are reminiscent of arthropod gap and Hox gene expression patterns (Fig. 7). It is thus possible that these genes contribute to body regionalization, especially in the anterior Hox-free part of the onychophoran body. 19

20 Figure 7. Gap and Hox gene-like expression patterns of Wnt genes. Dashed lines indicate additional segments. hl, head lobe; j, jaw; L1, first walking-limb-bearing segment; sp, slime papilla. Many of the Wnt genes are expressed in the limbs as they are in arthropods. But where in the limbs they are expressed varies between Euperipatoides kanangrensis and arthropods. In arthropods, several Wnt genes are expressed along the ventral side of the limbs. In Euperipatoides kanangrensis many of the Wnt genes are expressed in the tip of the limbs (Fig 8). Figure 8. Expression of Wnt genes in the limbs of E. kanangrensis. 20

21 Paper II The Frizzled receptors are part of the complex system in target cells that receive signaling Wnt ligands. The four subfamilies of Frizzled receptors have been characterized in a few species, which include the two ecdysozoan model species Caenorhabditis elegans and Drosophila melanogaster. Here we explore the frizzled genes in three panarthropod species: the spider Parasteatoda tepidariorum, the millipede Glomeris marginata and the velvet worm Euperipatoides kanangrensis. Each species has four frizzled genes, but Parasteatoda tepidariorum does not have a fz3 gene, but has two fz4 paralogs. Glomeris and Euperipatoides have representatives from all four subfamilies. Fz1 expression patterns in all the investigated species are consistent with a role for fz1 in segmentation across the panarthropods. Fz2 shows more variation throughout the species. Glomeris and Parasteatoda express fz2 in segmental stripes, which corresponds with the findings in Drosophila, which suggests that this member of the Fz family was involved in segmentation in the common ancestor of these animals. But Euperipatoides lacks segmental expression which states that it is unclear if this gene had a segmental role in the ancestor of panarthropods. All species express fz2 in the median head region suggesting that this is a conserved ancestral expression domain of this gene. Fz3 as said above was only found in Euperipatoides and Glomeris, the former expresses the gene in transverse segmental strips, as well as in the mouth and the anal valves. Glomeris expresses fz3 only in two dots in each eye field and in later stages in the anal valves. In Drosophila, the gene is expressed in segmental stripes, eye, leg discs and anal tissue. The ancestral role of fz3 is still unclear, but it could have a role in nervous system, eye and appendage development. The two paralogs of the Fz4 subfamily in Parasteatoda seem to have been subject to sub-functionalization, where some of the expression patterns exhibit similarities but there are differences as well. In comparison to the two other investigated species fz4 may be involved in nervous system development, segment formation in arthropods, as well as limb development among panarthropods. 21

22 Unpublished data My research now is focused on Priapulus caudatus and all the different gene families that play a role in body pattering and segmentation of segmented phyla. Hitherto investigated genes Eleven new Wnt genes were found in sequenced embryonic transcriptomes and a partially annotated sequenced genome (wg/wnt1 has already been published (Martín-Duran and Hejnol 2015)). The segment polarity genes, engrailed and hedgehog were obtained. Also the Notch gene and its receptor encoding gene Delta. We found all the ten known Hox genes (de Rosa et al. 1999), although recovered sequence information of three of them (Hox3, Deformed and Ultrabithorax) was too little for probe synthesis. Expression patterns of Wnt genes I have analysed the expression pattern of Wnt4, Wnt8 and Wnt16. At the introvert stage, all of them are expressed in the posterior pole of the embryo (around 5-7 days after fertilization) (Fig. 9). These expression patterns are comparable to the published expression pattern of wingless, and thus likely in the blastoporal region (Martín-Duran and Hejnol 2015). There is no expression pattern around the borders between the developing introvert and the trunk region, as I had assumed. 22

23 Figure 9. Gene expression in Priapulus caudatus embryos. Introvert stage (6-7 days old). Labial is expressed in the anterior part of the embryo except the very anterior cells. Wnt4, Wnt8 and Wnt16 are expressed in the posteriorly located blastoporal area. Expression patterns of Hox genes So far, I have analysed the expression pattern of labial, the most anterior Hox gene. As expected, labial is expressed in the anterior part of the embryo (Fig. 9). Interestingly, there is no expression of labial in the most anterior cells, which corresponds well with the expression of labial in arthropods (reviewed in Hughes and Kaufman 2002). Notably, the size of the region of the expression pattern varies which may not be explained by differences in developmental staging alone (Fig. 10). There is a variation in how far each embryo has developed in each stage-batch, but it should not be a huge variation. Figure 10. Expression of labial in Priapulus caudatus embryos of the introvert stage (6-7 days old). It shows the variation of the expression pattern of labial in similar stage-batches. 23

24 Future directions I am conducting my research on priapulids by investigating the expression pattern of the Wnt genes and the segment polarity genes, hedgehog and engrailed. The aim is to find out if these genes have anything to do with the border formation in priapulids. I also plan to look into the Notch pathway, which is another important and possibly conserved pathway in animal segmentation. I have also started to work on the Hox genes to get a general overview over the development of my research organism. 24

25 Acknowledgments I would like to thank a lot of people, first my supervisors, Graham Budd, Andreas Hejnol and Ralf Janssen. Especially Ralf who has encouraged me when nothing is working in the lab and for always pushing me to develop my skills in every aspect. I also want to thank all the people at the Sars Centre in Bergen and at the Sven Lovén centre in Fiskebäckskil for helping me collecting Priapulus caudatus and learning me useful techniques. Special thank also to Chema for all the help I received from him when I was in Bergen and all the help I got via . I also want to thank all the people in the Paleobiology program in Uppsala. I want to thank Johan and Gaëtan for all the pep talk on the lunches we have. Especially Johan who has listened to my complaints a dozen of times when nothing is working. I also want to thank my family for the support I got and all my friends who think that it is funny to have a friend who is working on penis worms. And last I want to thank my wife and daughter for the support and love. 25

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