A molecular view of onychophoran segmentation

Arthropod Structure & Development xxx (2016) 1e13

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A molecular view of onychophoran segmentation Ralf Janssen €gen 16, 75236 Uppsala, Sweden Uppsala University, Department of Earth Sciences, Palaeobiology, Villava

a r t i c l e i n f o

a b s t r a c t

Article history: Received 13 May 2016 Received in revised form 22 July 2016 Accepted 3 October 2016 Available online xxx

This paper summarizes our current knowledge on the expression and assumed function of Drosophila and (other) arthropod segmentation gene orthologs in Onychophora, a closely related outgroup to Arthropoda. This includes orthologs of the so-called Drosophila segmentation gene cascade including the Hox genes, as well as other genetic factors and pathways involved in non-drosophilid arthropods. Open questions about and around the topic are addressed, such as the definition of segments in onychophorans, the unclear regulation of conserved expression patterns downstream of non-conserved factors, and the potential role of mesodermal patterning in onychophoran segmentation. © 2016 Elsevier Ltd. All rights reserved.

Keywords: Onychophora Segmentation Development Euperipatoides

1. Introduction The onychophorans represent a small phylum of invertebrate animals closely related to the arthropods (e.g. Giribet and Edgecombe, 2012; Borner et al., 2014). Arthropods, the most successful of animal phyla, have conquered almost all habitats on our planet, and have a long fossil record dating back to the Cambrian. It is estimated that there are more than five million living arthropod species (e.g. Odegaard, 2000 and references therein). Two morphological features are believed to be responsible for this enormous success, their segmented anterior-posterior body axis and the evolution of segmented (jointed) limbs. This may be one of the reasons why the onychophorans are represented by a relatively small number of only around 200 described species (Oliveira et al., 2012). Onychophorans are not as clearly segmented as arthropods, lack tagmosis, and do not possess segmented limbs. This may have hampered their radiation and thus, in contrast to the arthropods with their countless morphological forms, all onychophorans are very similar, sharing a conserved morphology that has changed relatively little since the € m and Hou, 2001; Liu et al., 2008). DocCambrian (e.g. Bergstro umenting and finally understanding the differences between onychophorans and arthropods, especially with respect to body segmentation, is therefore in the spotlight of evolutionary developmental (EvoDevo) research.

Most insights about arthropod body segmentation, i.e. the genetic framework that controls it, come from groundbreaking studies on the model arthropod Drosophila melanogaster, a dipteran fly. Here, a hierarchic segmentation gene cascade patterns the early embryo step by step into the segments (reviewed in e.g. Akam, 1987; Pankratz and J€ ackle, 1993). Maternally provided effect genes (MEGs) function on top of this hierarchy. They form anteriorto-posterior and posterior-to-anterior protein gradients that control the expression of zygotically-expressed gap genes (GGs) in broad, multiple-segment wide, and partially overlapping domains €hnhofer and Nüsslein-Volhard, 1986; reviewed in McGregor, (Fro 2005). The GGs regulate the expression of pair-rule genes (PRGs) that are expressed in transversal stripes in alternating parasegments (J€ ackle et al., 1992). The interaction of PRGs is hierarchic itself, and so-called primary PRGs such as even-skipped and hairy control secondary PRGs, which partially control the tertiary PRG paired (Baumgartner and Noll, 1990). In a combinatorial mode the PRGs regulate the segment polarity genes (SPGs), which maintain the parasegment boundaries and define the segments' polarity (reviewed in e.g. Sanson, 2001) (Fig. 1). Finally, the Hox genes define segmental identity along the AP body axis (reviewed in Hughes and Kaufman, 2002a; Pick, 2015). Despite the simplicity of the segmentation gene cascade in Drosophila, this mode of segment formation (called long-germ developmental mode) only functions in embryos where all segments are patterned simultaneously from a pre-existing field of cells, the blastoderm (e.g. Sanson, 2001). This kind of development, however, is derived and is only found in some groups of higher

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Fig. 1. The Drosophila segmentation gene cascade (SGC) applied to Panarthropoda. Hox and segment polarity genes (SPG) are highly conserved in all Panarthropoda (also see Smith et al., 2016: Hox gene expression in Tardigrada). Function of tertiary pair-rule genes (tPRG) is more conserved than that of secondary PRG (sPRG) and that of sPRGs is more conserved than that of primary PRG (pPRG). Gap genes (GG) are less conserved in their function during segmentation than the bottom level genes (Hox, SPG, PRG), and finally maternal effect genes (MEG) are least conserved. Below the pyramid, presumed function of Drosophila segmentation gene orthologs (and additional segmentation genes) is summarized. Note that “Drosophila” represents also closely related species, Cyclorrapha, with the same mode of development and conserved segmentation mechanisms. Abbreviations and explanations: —, lack of function; -*1-, few orthologs with function; -*2-, localized maternal factors are present in some hexapods (Bucher et al., 2005); *, only few orthologs have been investigated (Eriksson et al., 2013a,b); hGG, head gap gene-like system; n.a., not applicable; n.i., not investigated; N/DL, Notch/Delta signaling; PES, posterior elongation system (see text for further information). Brackets indicate unclear situation or/and ongoing debate.

insects. In all other arthropods, including the majority of all hexapods, only the most anterior segments form from the blastoderm, and posterior segments are added in a single or double segment period from a posterior segment addition zone (SAZ) (Schoppmeier and Damen, 2005a; Janssen, 2011, 2014; Chipman et al., 2004; Davis and Patel, 2002) e the so-called short-germ developmental mode. Independent from the developmental mode, the expression and function of the arthropod Hox genes is highly conserved, although Drosophila has lost two of the canonical ten Hox genes, a number that is otherwise conserved among arthropods (e.g. Hughes and Kaufman, 2002a; Janssen and Damen, 2006; Manuel et al., 2006; Schwager et al., 2007; Pick, 2015; Serano et al., 2016). The same high level of conservation holds true for the arthropod SPG system (e.g. Tabata et al., 1992; Bejsovec and Wieschaus, 1993; Peterson et al., 1998; Abzhanov and Kaufman, 2000; Hughes and Kaufman, 2002b; Damen, 2002; Janssen et al., 2004; Simonnet et al., 2004;

Farzana and Brown, 2008; Janssen, 2012). The level at which the PRGs act, however, is less conserved, although investigation of PRG expression and function in arthropods revealed and/or suggested that most of the Drosophila PRG orthologs generally are involved in arthropod segmentation (e.g. Choe et al., 2006; Janssen et al., 2011a, 2012; Damen et al., 2005; Mito et al., 2007; Chipman and Akam, 2008; Eriksson et al., 2013a). The arthropod segmentation gene hierarchy appears to be only little conserved at the level of GGs and to even lesser extent at the level of MEGs (reviewed in e.g. Damen, 2007; Peel et al., 2005; McGregor, 2005). There are only few reports on GG expression and/or function of classical Drosophila GGs in non-insect arthropods (Schwager et al., 2009; Janssen et al., 2011b; Eriksson et al., 2013a), although other genes may act as gap genes in the early blastoderm (Pechmann et al., 2011). In insects, however, at least some of the GGs retained (i.e. had already evolved) their function in segmentation (Liu and Kaufman, 2004; Mito et al.,

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2006; Ben-David and Chipman, 2010; Liu and Patel, 2010; Blechert et al., 2011; Lavore et al., 2012; Nakao, 2015). Recent studies have shown that the mechanisms and gene interactions acting at the bottom level of the Drosophila segmentation gene cascade and which are also most conserved within Arthropoda appear to be conserved in onychophorans as well (Eriksson et al., 2009, 2010; Janssen and Budd, 2013; Janssen et al., 2014; Franke and Mayer, 2014; Franke et al., 2015) (summarized in Figs 1 and 7). The aim of this article is to summarize our current knowledge on onychophoran segmentation from a molecular (genetic) point of view. The available data on arthropod segmentation genes in onychophorans are analyzed and put into context. Since a thorough analysis of GG expression in onychophorans has not been conducted yet, such analysis is provided here for the first time. Furthermore, segmentation mechanisms that act in nondrosophilid insects and other arthropods are discussed. Patterning of the anterior-posterior axis on the level of segmental identity, as provided by the Hox genes, is reviewed, as well as the potential contribution of non-Hox genes in defining segmentidentity, especially with respect to the most anterior, Hox-free, region of the onychophoran (and arthropod) embryo. Finally, some open questions and problems concerning onychophoran segmentation are addressed here, such as the definition of the onychophoran segment, and the regulation of ‘conserved’ factors of the segmentation gene cascade in the absence of a conserved upstream acting gene regulatory network. 2. Methods 2.1. Embryo collection, fixation and developmental staging Embryos of the onychophoran Euperipatoides kanangrensis were prepared for in situ hybridization experiments, and staged as described in Janssen et al. (2010). 2.2. Gene cloning Total RNA was extracted (TRIZOL, Invitrogen, Carlsbad, CA) and reverse transcribed into cDNA (SuperscriptII first strand synthesis system for RT-PCR, Invitrogen). Gene fragments were isolated by amplification through RT-PCR using gene-specific primers (Table S1) based on a sequenced embryonic transcriptome. All fragments were subsequently cloned into pCR-II vectors (TA cloning kit dual promoter, Invitrogen) and sequenced by a commercial sequencing service (Macrogen, Seoul, Korea). Gene accession nos.: LT560240 (Ek-eg/knrl), LT560242 (Ek-Kr1), LT560243 (Ek-Kr2), LT560244 (Ek-tll), LT560239 (Ek-cnc), LT560241 (Ek-ems), LT560245 (Ek-unpg), and LN998118 (Ek-twi). 2.3. In situ hybridization Whole mount in situ hybridization was essentially performed as described in Janssen et al. (2015a, Supplementary data). Digoxigenin-labeled RNA probes were transcribed from the complete isolated gene fragments. Cell nuclei were stained by incubation at room temperature in approximately 2 mg/ml of the fluorescent dye 4-6-diamidin-2-phenylindol (DAPI) in phosphatebuffered saline with 0.1% Tween-20 (PBST) for ca. 20 min. 2.4. Data documentation All pictures were taken with a Leica DC100 digital camera under a Leica dissection microscope. The image processing software

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Adobe PHOTOSHOP CS2 (v. 9.0.1 for Apple Macintosh) was used for linear corrections of brightness, contrast and color values. 2.5. Gene identification Candidate genes were identified in the E. kanangrensis embryonic transcriptome database by reciprocal BLAST searches using arthropod sequences as queries. Single onychophoran orthologs of the diverged basic leucin zipper (bZIP) transcription factor (TF) cap-n-collar (cnc), the homeodomain containing TFs empty-spiracles (ems) and unplugged (unpg), the steroid receptor TF tailless (tll), the zinc finger (ZF) containing TF Krüppel (Kr) (two paralogs), and the basic helixloop-helix (bHLH) TF twist (twi) were identified that are significantly more similar to their arthropod orthologs than to any other gene in the well-annotated genomes of Drosophila and Tribolium. Therefore, no further phylogenetic analysis was conducted for these genes. The phylogenetic predictions are supported by the genes' conserved expression patterns in arthropods and the onychophoran. One single transcript was identified that shows significant sequence similarity to arthropod knirps-like genes (knirps, knirpsrelated, eagle (aka egon)) (Naggan Perl et al., 2013). Since the duplication of the ancestral gene likely happened in the insect lineage, the onychophoran ortholog, which is slightly more similar to eagle than to knirps-related was designated as eagle/knirpsrelated (Ek-eg/knrl). 3. Results 3.1. Analysis of gap gene orthologs in onychophorans A hunchback (hb) ortholog has recently been identified and its expression has been documented in great detail in a closely related onychophoran species, Euperipatoides rowelli (Treffkorn and Mayer, 2013). The expression pattern of E. kanangrensis hunchback has therefore not been investigated. Euperipatoides eagle/knirps-related (eg/knrl) is first expressed in the form of a broad domain in the head lobes posterior to the position of the frontal appendages, as well as in the frontal appendages, and weakly in the primordia of the limbs (Fig. 2A). This expression is later also visible in the outgrowing limbs (Fig. 2BeE). At later developmental stages eg/knrl is expressed strongly in the proximal part of the developing frontal appendages and in the head lobes (Fig. 2BeE). Expression in the other limbs is weaker but also restricted to proximal tissue. The most distal expression forms a ring in the slime papillae and the walking limbs, but not the jaws (Fig. 2E). Krüppel-1 (Kr1) is expressed transiently and in a salt-andpepper pattern in a few trunk segments, while the other segments both anterior and posterior to it do not express Kr1 at this point (Fig. 3A/B). This very specific pattern, however, is only seen in relatively young embryos. In younger embryos as the ones shown, as well as in older embryos, no expression was detectable. Kr2, is expressed in all tissues in the young embryo, but regions of higher expression are clearly recognizable in the head lobes including the primordia of the frontal appendages, and along the body axis where later the limbs form, and in tissue ventral to it (Fig. 3C). At later stages it becomes clear that this expression is restricted to the position of the limbs (including tissue ventral to the base of the limbs) (Fig. 3D/E). Euperipatoides tailless (tll) is expressed first at stage 9 in form of two domains in the head lobes (Fig. 4AeC). This anterior expression remains throughout development. At late stages tll is also

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Fig. 2. Expression of Euperipatoides eagle/knirps-related (eg/knrl). In all panels anterior is to the left. Stages are indicated. A Lateral view. Arrow and arrowhead mark enhanced expression in the anlage of the frontal appendage and the head lobe respectively. B Ventral view. Arrow and arrowhead as in A. C Lateral view. Same embryo as in B. D Lateral view. The asterisk marks lack of expression in the tip of the frontal appendages. E Dorsal view. Magnification of the head of the same embryo as shown in D. Arrows point to a ring of expression in the limbs. A0 eD0 : DAPI counterstained embryos as shown in AeD. Arrows, arrowheads and asterisk are in same position in bright field and DAPI-stained embryos. Abbreviations: fap, frontal appendage; hl, head lobe; L1, first walking limb; sp, slime papilla.

expressed around the mouth and the anterior domain splits into two (Fig. 4D). A possible ortholog of E. kanangrensis giant could not be identified (summarized in Supplementary Table S2). 3.2. Analysis of so-called head gap gene orthologs The embryonic expression of E. kanangrensis orthodenticle (otd) has been described previously in Steinmetz et al. (2010) and Eriksson et al. (2013b). The expression of E. kanangrensis collier (col) has been described in Janssen et al. (2011c). A possible ortholog of E. kanangrensis buttonhead (btd) could not be identified. The onychophoran cap-n-collar (cnc) gene is ubiquitously expressed at low levels throughout embryogenesis (embryos of the stages 3e20 have been investigated; data not shown). Euperipatoides empty-spiracles (ems) is expressed in a segmental pattern (Fig. 5A/B). The most anterior expression of ems is at the anterior border of the jaw-bearing segment; the head lobes do not express ems. Within the segments where ems is expressed, this early expression covers the anterior of the limb primordia and the tissue anterior to that. This becomes obvious in

later stages when the limbs have been grown out already (Fig. 5B). At these later stages, a new expression domain appears in the posterior of the limbs, resulting in an anterior and a posterior domain within all appendages (except the frontal appendages). Another segmental expression spans the tissue between the bases of the limbs (Fig. 5B). 3.3. Expression of the nervous system-patterning gene unplugged At early stages, unplugged (unpg) is ubiquitously expressed, except for tissue anterior to the middle of the jaw-bearing segment. At later stages, expression in the jaw-bearing segment is stronger than in the other, more posterior, segments (Fig. 5C/D). 3.4. Expression of the mesodermal marker twist Euperipatoides twist (twi) is strongly expressed in a dynamic pattern in the posterior pit region, and later also in the segment addition zone except for its most anterior region that is adjacent to the mouth-anus furrow (Fig. 6). At some developmental stages it is also weakly expressed in the last set of newly formed somites (e.g. Fig. 6B).

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Fig. 3. Expression of Euperipatoides Krüppel-1 (Kr1) and Krüppel-2 (Kr2). In all panels anterior is to the left. Stages and gene are indicated. A Ventral view. Red bars mark border of expression. B lateral view. Bars as in A. Note that the domain of expression is now shifted towards posterior by a few segments. C Lateral view. The arrow marks expression in the primordium of the frontal appendage. D Lateral view. Arrow as in C. E Lateral view. A0 eD0 : DAPI counterstained embryos as shown in AeD. Arrows and bars are in identical position in bright field and DAPI-stained embryos. Abbreviations as in Fig. 2; bp, blastoporal region; L2/6, second and sixth walking limb-bearing segment respectively.

Fig. 4. Expression of Euperipatoides tailless (tll). In all panels anterior is to the left. Stages are indicated. A Ventral view. Arrowhead marks upcoming expression in the head lobes. Asterisk marks a second domain of expression in the head lobes. B Ventral view. Asterisk and arrowhead as in A. C Lateral view. Asterisk and arrowhead as in A. D Lateral view. Asterisk and arrowhead as in A. Arrow points to a third domain of expression within the head lobes. Note expression around the mouth (m). A0 eC0 : DAPI counterstained embryos as shown in AeC. Arrowheads and asterisks are in identical position in bright field and DAPI-stained embryos. Abbreviations as in Fig. 2; m, mouth.

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Fig. 5. Expression of Euperipatoides unplugged (unpg) and empty-spiracles (ems). In all panels anterior is to the left. Stages and gene are indicated. A Lateral view. The arrow marks the anterior border of expression. B Ventral view. Arrow as in A. C Lateral view. Arrow as in A. D Lateral view. A0 eC0 : DAPI counterstained embryos as shown in AeD. Arrows are in identical position in bright field and DAPI-stained embryos. Abbreviations as in Fig. 2; j, jaw-bearing segment.

4. Discussion 4.1. The role of Drosophila segmentation genes in onychophoran segmentation 4.1.1. Hox genes In a combinatorial mode, the Hox genes provide positional information along the anterior-posterior body axis, and give each segment its identity (e.g. Akam, 1998; Lawrence and Morata, 1994). There are ten ancestral arthropod Hox genes (reviewed in Hughes and Kaufman, 2002a), and this number is indeed conserved in onychophorans as well (Grenier et al., 1997). The expression patterns of all ten onychophoran Hox genes have recently been studied (Eriksson et al., 2010; Janssen et al., 2014) showing the typical conserved patterns. All onychophoran Hox genes posses a clear anterior border of expression and are expressed throughout development in all segments posterior to this border. One exception, however, is the expression of the eighth Hox gene, Ultrabithorax (Ubx) that forms a posterior to anterior gradient (Janssen et al., 2014). The idea was put forward that Ubx could be responsible for the definition of posterior segments, which in the onychophoran express the same set of Hox genes, by providing different amounts of its gene product (Janssen et al., 2014).

4.1.2. Segment polarity genes (SPGs) In Drosophila, activation of SPGs by the upstream acting pairrule genes (PRGs) results in the establishment and maintenance of the parasegment boundaries, and also establishes anteriorposterior polarity of the segments (e.g. Sanson, 2001). Like the Hox genes, the level of SPGs is highly conserved in arthropods, and so is the set of genes involved (e.g. Damen, 2002; Simonnet et al., 2004; Janssen et al., 2008; Farzana and Brown, 2008). engrailed (en) and hedgehog (hh) are expressed posterior in the segment, while wingless (wg), patched (ptc) and cubitus-interruptus (ci) are expressed anterior adjacent to the domain of en/hh expression. Notum, a modifier of Wg function is co-expressed with wg, but is not considered a classical SPG (Giraldez et al., 2002). Another factor involved is H15 (aka midline (mid)), which is expressed anterior to en/hh as well where it breaks the symmetry of Hh-dependent activation of wg expression (Buescher et al., 2004). In onychophorans, the network of SPG interaction appears to be highly conserved as well, although there are some important differences (Eriksson et al., 2009; Janssen and Budd, 2013; Franke and Mayer, 2014). Firstly, H15 is not expressed in a segmental fashion in the onychophoran E. kanangrensis (Janssen et al., 2015a), and secondly, the domain of en-expression is broader than that of its target gene hh (Janssen and Budd, 2013; Franke and Mayer, 2014). The latter causes some confusion about the whereabouts of the (para)

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Fig. 6. Expression of Euperipatoides twist (twi). In all panels anterior is to the left. Ventral views. Stages are indicated. A Arrow marks anterior border of expression within the posterior pit region. B Arrowhead marks anterior border of expression in newly formed somites. C Asterisks mark expression at the border where the new somites are forming. D Asterisks as in C. A0 eD0 : DAPI counterstained embryos as shown in AeD. Arrows, arrowheads and asterisks are in same position in bright field and DAPI-stained embryos. Abbreviations as in Fig. 2; a, anus; m, mouth; m-a, mouth-anus furrow; saz, segment addition zone.

segmental boundaries in onychophorans compared to arthropods (discussed below). In conclusion, like the Hox genes, SPG interaction is highly conserved in arthropods and onychophorans, suggesting that these two genetic components were part of the ancestral panarthropod body segmentation network. 4.1.3. Pair-rule genes (PRGs) The role of the PRGs is less conserved, which is understandable given the relatively earlier expression/function of these genes when patterning the Drosophila blastoderm. These genes cannot be expressed in the same double-segmental pattern as it is the case in Drosophila, because in short-germ arthropods posterior segments are added gradually. However, PRG orthologs appear to be generally involved in segmentation as they are (often) expressed in segmental stripes in newly formed posterior segments, or are expressed within the SAZ from which these segments are generated (e.g. Damen et al., 2000; Janssen et al., 2011a). The internal hierarchy within the PRG-system as present in Drosophila appears, based on the available functional and geneexpression data, to be conserved in arthropods as well, although some of the genetic players may have swapped position within the hierarchy (e.g. Chipman et al., 2004; Choe et al., 2006; Chipman and Akam, 2008; Janssen et al., 2011a). Gene expression analysis of PRG orthologs in onychophorans revealed that those PRGs that act high in the arthropod PRGhierarchy likely are not involved in segmentation (with the possible exception of even-skipped (eve)) (Janssen and Budd, 2013).

Genes that act downstream of the arthropod primary PRGs, however, are expressed in segmental patterns which may point to a conserved (or similar) function in segmental patterning (rather than segment formation) (Janssen and Budd, 2013). Interestingly, the onychophoran paired (prd) gene (aka Pax3/7, pby), a gene that is sometimes referred to as a tertiary PRG because it is partially under control of secondary PRGs in Drosophila, is expressed in a highly conserved pattern in arthropods as well as in onychophorans (Janssen and Budd, 2013; Franke et al., 2015). This is likely because it directly interacts with the highly conserved SPG network (discussed above). 4.1.4. Gap genes (GGs) The level of GGs in arthropods is even less conserved than the PRG-system (reviewed in e.g. Damen, 2007; Peel et al., 2005) with only few reports on GG expression and/or function of classical Drosophila GGs outside the insects. These reports are restricted to a single gene, hunchback (hb) a GG that in Drosophila is also involved in anterior patterning, both functioning as a GG as well as a maternal effect gene (MEG) (discussed below) (Schwager et al., 2009; Janssen et al., 2011b; Eriksson et al., 2013a,b). Generally, the anterior gap gene-like system (including genes such as hb, orthodenticle (otd), empty-spiracles (ems) and buttonhead (btd)) that is active in the early blastoderm is more conserved than the Drosophila trunk GG-system. Hitherto, the GGs have been investigated only very poorly in onychophorans, most probably because of their assumed low grade of conservation and thus influence on segment formation. A single

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publication deals with the expression analysis of a single GG, hb, and there is no report on an expression profile of hb that would suggest a function in segmentation (Franke and Mayer, 2014). Another article describes the expression of collier (col) a member of the anterior patterning network, but again there is no sign of expression that would suggest a special role in anterior patterning (Janssen et al., 2011c). The analysis of trunk GG orthologs and GG-like head patterning genes revealed that none of these genes are involved in onychophoran segmentation (Figs. 2e5). The trunk GG ortholog gt is either missing, or is not active in ontogenetic stages, and expression of Kr1, Kr2, eg/knrl and tll suggest a function in nervous system (Kr1þKr2) and late head/brain development (eg/knrlþtll). These functions have also been reported for these orthologs in ar€der et al., 2000; Stollewerk et al., 2003a; Liu and thropods (Schro Kaufman, 2004; Cerny et al., 2008; Mito et al., 2006; Wilson et al., 2010; Janssen et al., 2011b; Peel et al., 2013; Weisbrod et al., 2013; Guillermin et al., 2015; this study) (Supplementary Figs. S1 and S2), and it is thus likely that their function in neurogenesis and head patterning are conserved. Analysis of head gap genes revealed the likely absence of buttonhead (btd), ubiquitous expression of cnc (not shown), and no role of col, ems and hb in head patterning (Janssen et al., 2011c; Franke and Mayer, 2014; this study) (Fig. 5). Although otd, another conserved anterior head GG-like factor is expressed early during development and specifically in the onychophoran head lobes (Steinmetz et al., 2010; Eriksson et al., 2013a), this is likely correlated to a conserved anterior region present in all bilaterians (e.g. Steinmetz et al., 2010). Conclusively, it appears that there is neither a conserved head GG-like patterning system in onychophorans, nor a trunk GG system. However, an interesting feature is the temporal expression of Kr1 in a restricted subset of anterior trunk segments in E. kanangrensis. This pattern is somewhat reminiscent of classical GG expression and what is seen in some short germ arthropods as well (Schoppmeier, 2003; Janssen et al., 2011b); thus Kr1 may represent a proto-GG that evolved a function as bona fide GG later in evolution (Peel and Akam, 2003).

4.1.5. Maternally provided effect genes (MEGs) In Drosophila, the MEGs function in a syncytial blastoderm, where they are initially located at the poles of the egg, and from where they form gradients of mRNA or protein from anterior to posterior and posterior to anterior respectively. Anterior determinants are bicoid (bcd) and hunchback (hb), whereas posterior determinants are nanos (nos) and caudal (cad). In a concentrationdependent way these genes control their downstream targets, mainly the GGs (e.g. Lawrence, 1992). In long-germ arthropods, this simple system cannot function and recent work has shown that, although some factors are principally conserved, major differences occur in posterior and even anterior patterning in short-germ in€der, 2003; Bucher and Klingler, 2004; Bucher et al., sects (Schro 2005; Choe et al., 2006). Unsurprisingly, bcd was not found in the sequenced onychophoran transcriptome, and neither was nos (somewhat surprisingly). Although hb is present, there is no sign of it being localized at either pole of the embryo (see Eriksson and Tait, 2012 or Janssen et al., 2015b for an overview over early development), or forming any kind of gradient (Treffkorn and Mayer, 2013). Interestingly, however, the conserved posterior factor cad is not only expressed at the posterior pole of the developing onychophoran embryo, but it also forms some sort of a short range posteriorto-anterior gradient, at least in some early developmental stages (Janssen and Budd, 2013; Janssen et al., 2015b).

4.2. Definition of the onychophoran ‘segment’: a “groovy” story In Drosophila, the parasegments are defined by the expression of SPGs such as engrailed (en) and wingless (wg) with the parasegmental border (and its associated groove) located exactly where the expression domains of these two genes meet (e.g. Martinez Arias, 1993; Larsen et al., 2008). The segmental borders, the morphologically visible indentation of the adult arthropods, form at the posterior edge of en expression (Martinez Arias, 1993; Larsen et al., 2003). Since in arthropods the expression domains of en and its target hedgehog (hh) are identical, either en or hh can serve as (para)segmental markers (e.g. Janssen et al., 2004; Simonnet et al., 2004). Given that the expression patterns of the SPGs are highly conserved in arthropods (discussed above), expression of wg can be used to define (para)segmental boundaries as well. In onychophorans, however, the situation is different, since here expression of en and hh is not identical with regard to their posterior extension, although they share a common anterior border of expression (Janssen and Budd, 2013; Franke and Mayer, 2014). It is thus, from a molecular point of view, unclear if boundaries (if present at all) are defined by hh- or en-expression. Since the border between wg/patched (ptc) expressing cells and en/hh expressing cells is conserved in onychophorans and arthropods, parasegmental organization may be conserved. In arthropods, the later formation of segmental ectodermal indentations would easily solve such dilemma, but in onychophorans such segmental grooves do not form. Franke and Mayer (2014), however, argue in their recent paper against parasegmental organization. They claim that embryonic segmental grooves exist and that the location of SPG expression relative to this “grooves” does, according to these authors, not change during ontogeny, although this is to some extent a characteristic of arthropod segmentation. They provide five lines of evidence supporting their hypothesis. 1) Identification of morphologically visible grooves posterior to segmental expression of hh, 2) temporal differences between boundary formation and SPG expression, 3) diffuse boundaries of SPG expression rather than sharp borders as found in arthropods, 4) expression of Hox genes in a segmental, rather than a parasegmental pattern, 5) neuroanatomical evidence (discussed in Franke and Mayer, 2014). Consequently, they conclude that assuming a common origin of segmentation in annelids and arthropods is premature, because the parasegmental organization as the uniting factor would be missing (e.g. Dray et al., 2010). Although the discussion of the origin of segmentation is not topic of this paper, the line of argumentation put forward by Franke and Mayer (2014) demands critical reevaluation. Firstly, there is no clear evidence that the “grooves” described by Franke and Mayer (2014) are homologous to arthropod segmental or parasegmental grooves. Although the authors claim that these “grooves” are not the result of the underlying mesoderm, they do not deliver any evidence for this assumption, and thus, it remains likely that the grooves actually are the consequence of somite formation. In short, the assumption that there are no homologous grooves would render the argument of “delayed” expression invalid. To interpret the anterior borders of SPG expression in onychophorans as diffuse is questionable as well, based on the available data (see data in Eriksson et al., 2009; Janssen and Budd, 2013; Franke and Mayer, 2014). Finally, the Hox gene argument is not convincing either, because a similar distribution of segmental and (para?)segmental anterior borders has been described for various Hox genes in various arthropods (Janssen et al., 2014 and references therein). It appears more plausible that the “grooves” seen in onychophoran segmentation are a secondary product of the

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underlying mesodermal blocks, the somites (Fig. S3). This would explain the formation of the “grooves” prior to the onset of SPG expression, and why there is no evidence of cells that sink in or become bottle-shaped as seen in Drosophila, not even temporarily (cf. Larsen et al., 2003) (Fig. S3). In support of this theory, the onychophoran mesodermal segmental blocks form early and from the posterior located blastoporal region (e.g. Manton, 1949; Eriksson and Tait, 2012; Janssen et al., 2015b), and while they mature (grow in size), the “grooves” appear. From a molecular point of view, the parasegmental organization of ectodermal patterning is thus likely conserved in arthropods and in onychophorans (even though convergent evolution can not be ruled out), as reflected by the conserved expression pattern of most of the investigated SPGs and associated factors. It remains unclear, however, where the segmental border in onychophorans is located (if existing at all), either posterior of the en-domain, or as in arthropods, posterior of the en/hh-domain. In any case, the molecular differences in en and hh patterning of the ectoderm do not cause any problems for the onychophoran, or in other words, do not require further debate. Since segmental grooves do not form in onychophorans, the genetic network underlying groove formation in arthropods like Drosophila (Larsen et al., 2003) can be different without any correlated consequences. Whether this represents an ancestral panarthropod character, or if groove/segment formation has been lost in onychophorans remains unclear until future investigation in tardigrades may (depending on the phylogenetic position of onychophorans relative to tardigrades) answer this question. 4.3. What controls the conserved patterns of the SPGs? Strikingly, in onychophorans, the higher-level hierarchy of the PRG-system, as a likely regulator of SPGs and lower-level PRGs, is not conserved (discussed above). This raises the question how the conserved expression patterns of the latter two classes of segmentation genes are regulated in their distinct patterns. One molecular component that is possibly part of the ancestral arthropod segmentation mechanisms is Delta/Notch (Dl/N) signaling, although there is an ongoing debate about this topic (e.g. Stollewerk et al., 2003b; Schoppmeier and Damen, 2005b; Dove and Stollewerk, 2003; Chesebro et al., 2012; Eriksson et al., 2013a). However, gene expression analysis of N, Dl, and other associated factors has shown, that Dl/N signaling is likely not involved in onychophoran segmentation (Janssen and Budd, 2016). If at all, expression of these genes suggests a function in posterior elongation, another conserved role of Dl/N signaling. As discussed in Janssen and Budd (2016), in onychophorans, all components of the arthropod posterior elongation network appear to be in place, even-skipped (eve), caudal (cad), as well as (in general) Wnt signaling, and the posterior Hox genes. However, despite the fact that the posterior elongation network is in place, this does not (necessarily) solve the problem of how to regulate conserved segmental patterning of the SPGs and secondary/tertiary PRGs (Eriksson et al., 2009; Janssen and Budd, 2013; Franke and Mayer, 2014; Franke et al., 2015) 4.4. Ectodermal vs mesodermal segmentation Segmentation in arthropods is predominantly seen in the ectoderm. This is especially obvious in Drosophila where the anterior-posterior body axis is patterned prior to gastrulation and the formation of mesoderm. While the scenario in Drosophila with its special mode of development may not be representative, studies in short-germ arthropods support the hypothesis that body

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segmentation is controlled by the ectoderm, and that signals from there cause segmentation of the underlying mesoderm, at least in insects and crustaceans (Bock, 1941; Haget, 1953; Hannibal et al., 2012; summarized in Scholtz and Wolff, 2013). The hypothesis that ectodermal segmentation is an ancestral feature of Panarthropoda, however, is not universally accepted. Budd (2001) suggests a different scenario in which mesodermal segmentation is ancestral, and thus, ectodermal segmentation a secondary trait. His hypothesis rests on the finding that obvious arthropod ancestors as revealed by the fossil record (e.g. Budd, 1996, 1999; Smith and Ortega-Hernandezm, 2014) possess an unsegmented ectoderm, but a segmented mesoderm (Budd, 2001). In Budd's hypothesis these ancestors, initially equipped with short bumpy limbs, evolved longer limbs, which then demanded for spatial reasons synchronized control. This control was, as suggested by Budd (2001) achieved by the evolution (or use) of ectodermal epidermal plates that could serve as muscle attachment sides. Thus, according to Budd (2001) patterning of the ectoderm evolved secondary and was induced by the evolutionary older mesodermal patterning. A second line of evidence came from the finding that the number of mesodermal teloblasts (stem cells that form the segments) in malacostracan crustaceans is highly conserved, while the number of ectodermal teloblasts varies, or is completely replaced by other mechanisms. Scholtz (1990) argued based on this fact that the conservation of mesoderm segmentation must be critical and would possibly provide an inductive signal for the segmentation of the ectoderm. Since the available data supporting predominant ectodermal segmentation are restricted to insects and crustaceans, it remains an open question if this represents a conserved and ancestral character of (Pan)arthropoda, or if it represents a pancrustacean novelty. This leads back to another unresolved issue that is discussed above: how to achieve a conserved segmental pattern as represented by the expression of the SPGs and the tertiary PRG prd in the absence of an obvious upstream acting patterning system? Typical arthropod segmentation genes that function upstream of the SPGs (i.e. N/Dl, primary PRGs) are expressed in the SAZ or are cycling through the SAZ and newly formed segments. Interestingly, in the onychophoran none of these genes cycle or show any expression that would suggest a function in segment patterning (Janssen and Budd, 2013, 2016). However, one gene appears to be expressed in a dynamic pattern in the SAZ and is clearly involved in the formation of the segmental mesodermal blocks, the somites (Fig. 6). This gene is the highly conserved mesodermal master gene twist (twi) (e.g. Leptin, 1991; Yamazaki et al., 2005; Handel et al., 2005; Price and Patel, 2008; Pfeifer et al., 2013; Passamaneck et al., 2015; Martin-Duran and Hejnol, 2015). It could thus be that there exists indeed a mesoderm-controlled mechanism of ectodermal patterning in onychophorans. The currently available data on twist expression, however, are of rather preliminary nature. It is indeed unclear if the ‘dynamics’ are due to cell movement or cycling (a wave of transcriptional activity that is transferred from one cell to another). If the former holds true, the pattern could merely be the result of the morphological formation of the somites, and thus a secondary signal rather than an inductive signal for the patterning of the ectoderm. 4.5. Patterning of the Hox-free anterior head In arthropods, the anterior head is free from Hox expression and thus is patterned in a Hox-independent way. The same holds true for the onychophorans (Eriksson et al., 2010; Janssen et al., 2014). This raises the question how the anterior of the onychophoran

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head, i.e. all segments anterior to the slime papillae-bearing segment is patterned. A recent analysis of Wnt gene expression in E. kanangrensis revealed some unusual patterns in broad multiple segment-wide domains early during development (Hogvall et al., 2014). The anterior border of two of the Wnt genes Wnt2 and Wnt4 is located more anterior than the most anterior Hox pattern, i.e. in the anterior of the jaw-bearing segment and the posterior half of the head lobes respectively (Hogvall et al., 2014) (summarized in Fig. 7). Another Wnt gene, Wnt5, is expressed in the same domain as the Hox gene Deformed (Dfd), suggesting that Wnt5 is either under control of Dfd, or that Wnt5 is involved in the regulation of Dfd. Either way, the anterior expression of Wnt2 and Wnt4 cannot be controlled by Hox patterning and is either

instructive for anterior patterning, or under control of a hitherto unknown anterior patterning system. Gene expression analysis revealed that two more genes are possibly involved in anterior patterning and are expressed in overall patterns that resemble that of the Hox genes, i.e. a sharp anterior border of expression and expression in all tissue posterior to that. These genes are ems and unpg (Figs 5 and 7). The anterior border of expression of both genes lies anterior to that of the Hox genes and is therefore rather unique, suggesting a specific function in anterior patterning. This function is likely ancestral since these patterns are conserved in arthropods as well (Chiang et al., 1995; Walldorf and Gehring, 1992; Simonnet et al., 2006; Schinko et al., 2008; Birkan et al., 2011; this study) (Fig. S2).

Fig. 7. Summary of Hox gene expression and potential segment-identity defining genes in Euperipatoides. On top: last embryonic stage of Euperipatoides kanangrensis (close to birth). Abbreviations: 1e15, number of walking limb; abdA, abdominal-A; AbdB, Abdominal-B; Antp, Antennapedia; Dfd, Deformed; e, eye; ems, empty-spiracles; fap, frontal appendage; ftz, fushi-tarazu; L1, first walking limb; lab, labial; m(j), mouth (with internal jaws (j)); pb, proboscipedia; saz, segment additional zone; Scr, Sex combs reduced; sp, slime papilla; Ubx, Ultrabithorax; unpg, unplugged.

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Acknowledgements This work has been supported by the Swedish Research Council. I gratefully acknowledge the support of the New South Wales Government Department of Environment and Climate Change by provision of a permit SL100159 to collect onychophorans at Kanangra-Boyd National Park and to the Australian Government Department of the Environment, Water, Heritage and the Arts for export permits WT2009-4598 and WT2012-4704. I would like to thank Glenn A Brock, David Mathieson, Robyn Stutchbury, and especially Noel Tait, for their help during onychophoran collection. Nico Posnien and Alistair McGregor helped with the analysis of the onychophoran transcriptome, and Graham E Budd is thanked for proofreading of the final version of the manuscript.

Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.asd.2016.10.004

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Please cite this article in press as: Janssen, R., A molecular view of onychophoran segmentation, Arthropod Structure & Development (2016), http://dx.doi.org/10.1016/j.asd.2016.10.004