Analysis of the Wnt gene repertoire in an onychophoran provides new insights into the evolution of segmentation
© Hogvall et al.; licensee BioMed Central Ltd. 2014
Received: 16 January 2014
Accepted: 11 March 2014
Published: 3 April 2014
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.
KeywordsDevelopment Evolution Segmentation Segment polarity Wnt signalling
The phylum Onychophora is represented by only around 200 described species . Like their probable sister group, the arthropods, onychophorans are segmented, a fact that is most obvious from the arrangement of up to 43 pairs of walking limbs on these animals. However, onychophorans differ from arthropods because they lack intersegmental ectodermal grooves, tagmosis is absent and their limbs are unsegmented . Despite the great interest in, and growing understanding of, all aspects of arthropod biology, including the genetic regulation of segmentation (reviewed in, for example, [3–7]), relatively little is known about the onychophorans.
In Drosophila melanogaster, segmentation is under control of a hierarchic segmentation gene cascade that initially transforms aperiodic patterns of genetic information along the anterior-posterior body axis into a periodic pattern [8, 9]. Comparative studies have revealed that at least some components of this hierarchical network are conserved and that the function of segment polarity genes in particular has been maintained during the evolution of arthropods and onychophorans [10–18]. The segment polarity genes act later in the hierarchy downstream of maternal effect genes, gap genes and pair rule genes, and regulate segment polarity and maintain segmental boundaries. The network of segment polarity genes includes morphogens, such as Hedgehog (Hh) and Wingless (wg/Wnt1).
The Wnt gene family comprises 13 subfamilies, of which 12 are found in protostomes, with Wnt3 having been lost in the lineage leading to these animals [19, 20]. Preliminary studies of the Wnt gene repertoire in arthropods suggest some lineages have lost one or more Wnt genes in the course of evolution (summarized in ): for example, Wnt2 and Wnt4 appear to have been lost in insects.
To further study the role that Wnt genes play in development and evolution, we surveyed the repertoire of these genes in the onychophoran Euperipatoides kanangrensis and investigated their expression during its embryogenesis. We found that at least 11 of the predicted 12 Wnt genes found in protostomes are expressed during onychophoran ontogenesis. Our data suggest that onychophoran Wnt genes are likely to be involved in segment border formation or maintenance, intrasegmental patterning and possibly even the determination of segment identity. The latter function would not only represent an onychophoran-specific feature of Wnt gene function, but also suggest a role for these genes in segmentation beyond that of segment regionalization.
Animal husbandry and embryo preparation
Female specimens of E. kanangrensis were collected in Kanangra-Boyd National Park, New South Wales, Australia. To obtain all developmental stages, we dissected developing embryos of various stages in the months from September to December. Each female carries up to 100 embryos, representing a series of developing stages (sometimes even ranging from the one-cell stage up to the fully developed embryo). The chorion and vitelline membrane were removed by hand with Dumont size 5 forceps and directly afterwards the embryos were fixed in 4% formaldehyde in 0.1 M phosphate-buffered saline with 0.1% Tween-20 (PBST) (pH 7.4) for four to six hours at room temperature. Embryos were then dehydrated stepwise into 100% methanol and stored at −20°C for at least three weeks before being used for in-situ hybridization experiments.
PCR and gene cloning
RNA isolation and cDNA synthesis were described in . Gene fragments of all Wnt gene orthologues described here were isolated by means of PCR with gene specific primers based on the sequences found in a sequenced embryonic transcriptome. For further information on the transcriptome see .
All Wnt gene fragments were cloned into the pCRII vector (Invitrogen, Carlsbad, CA, USA), and sequences were determined by means of Big Dye chemistry on an ABI3730XL analyzer by a commercial sequencing service (Macrogen, Amsterdam, The Netherlands). Sequences of the newly discovered E. kanangrensis Wnt genes are available from the EMBL nucleotide database under accession numbers HG529208 (Wnt2), HG529209 (Wnt4), HG529210 (Wnt5), HG529211 (Wnt6), HG529212 (Wnt7), HG529213 (Wnt9), HG529214 (Wnt10), HG529215 (Wnt11), HG529216 (Wnt16), and HG529217 (WntA).
In-situ hybridization, cell nuclei staining and data documentation
In-situ hybridization experiments were performed as described previously . Cell nuclei were stained with 1 μg/ml DAPI (4-6-diamidino-2-phenylindole) in PBST for 20 minutes followed by several washing steps in PBST. Embryos were analyzed under a Leica dissection microscope equipped with a Leica DC100 digital camera. Brightness, contrast and colour values were adjusted if necessary, using the image processing software Adobe Photoshop CS2 (Version 9.0.1 for Apple Macintosh).
Bayesian phylogenetic analyses were performed with MrBayes  using a fixed WAG 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. The final topology was estimated using 1,100,000 cycles for the 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.
The Wnt gene repertoire of E. kanangrensis
Expression of E. kanangrensis wg/Wnt1
The expression of the E. kanangrensis wingless (wg/Wnt1) orthologue has been described previously . It is expressed like a typical segment polarity gene in transverse stripes in the middle of each segment and anterior to the expression of engrailed (en) and hedgehog (hh) , as well as in the tips of all developing appendages .
Expression of E. kanangrensis Wnt2
Expression of E. kanangrensis Wnt4
Expression of E. kanangrensis Wnt5
Expression of E. kanangrensis Wnt6
Expression of E. kanangrensis Wnt7
Expression of Wnt7 is also absent from early developmental stages (not shown). The earliest expression is observed at the bases of all the appendages of the trunk segments (Figure 7E). At subsequent stages, this expression becomes stronger and eventually transforms into a series of short longitudinal stripes in the mesoderm between the bases of the limbs along the anterior-posterior axis of the embryo (Figure 7F, G). During late stages, segmental expression appears in the developing ventral nervous system (Figure 7H, I).
Expression of E. kanangrensis Wnt9
Expression of E. kanangrensis Wnt10
During early stages, Wnt10 is weakly expressed throughout E. kanangrensis embryos (not shown). Later, differential expression appears in the tips of the limb buds and as transverse stripes in the centre of the trunk segments (Figure 8G-J). However, expression in the tips of the frontal appendages is weaker than in the other appendages (Figure 8I). Segmental stripes of expression are located posteriorly adjacent to the openings of the salivary glands, which also express Wnt10 (Figure 8J).
Expression of E. kanangrensis Wnt11
Expression of E. kanangrensis Wnt16
Expression of E. kanangrensis WntA
Wnt genes and segment identity
Since the composition of the anterior head in onychophorans is still uncertain, the anterior border of Wnt2 expression within the head lobes indirectly raises the question of whether the head lobes represent two segments (or at least two independently patterned regions). So far, only one coelomic pouch has been identified in the developing head lobes (for example, ), and only one transverse stripe of hedgehog (hh) expression lies at the posterior rim of the head lobe .
Wnt genes in segmentation and segment regionalization
The arthropod wingless/Wnt1 (wg/Wnt1) gene is a classical segment polarity gene that is involved in maintaining segmental boundaries and intrasegmental patterning (reviewed in [32–34]). In D. melanogaster, other arthropods, and onychophorans, wg is expressed anterior adjacent to the expression of engrailed (en) (for example, [35, 36, 14, 17]).
The onychophoran segment polarity gene orthologues are first expressed in their typical pattern in adjacent transverse stripes at around stage 10 [17, 18]; note that en is expressed considerably earlier. However, most of the Wnt genes are expressed in segment polarity gene-like patterns only at later stages. The temporal delay of segmental patterning in comparison with segment polarity genes, such as en, wg/Wnt1 and hh[17, 18], suggests that onychophoran Wnt genes are not generally involved in the determination of (morphologically invisible) intersegmental boundaries, but that their function is rather restricted to intrasegmental patterning.
All onychophoran segments are added from the posterior pit region, the SAZ. Since some of the Wnt genes are expressed in the SAZ, these genes may be involved in segment addition. The involvement of Wnt genes during segmentation has been demonstrated by the knocking down of components of the canonical Wnt gene network in insects [37–41]. In the basally branching insect Periplaneta americana, and in the spider Parasteatoda tepidariorum, the function of wg/Wnt1 and Wnt8, respectively, are indeed crucial for posterior segment addition [42–44]. In E. kanangrensis, we find that all Wnt genes except Wnt6 and Wnt7 are expressed in the SAZ. Of these, wg/Wnt1, Wnt2, Wnt4, Wnt5, Wnt11 and Wnt16 are all expressed prominently in the SAZ. Others, such as Wnt9, Wnt10 and WntA are only weakly expressed in the SAZ, or are expressed ubiquitously at some stages and thus also in the SAZ. Overall, these expression patterns suggest a function of onychophoran Wnt genes during segment addition, similar to that reported for insects and a spider.
Conserved and diverged aspects of Wnt expression in arthropods and onychophorans
Wnt1/wg is the best studied arthropod Wnt gene (reviewed in [45, 46]). Its expression in transverse segmental stripes anterior adjacent to engrailed (en) is principally conserved in all arthropods [12, 11, 14], and even in E. kanangrensis[17, 18]. The segment polarity gene-like function, however, has only been directly demonstrated for some insects [38, 39, 47].
Expression of Wnt2 has been studied in the millipede Glomeris marginata, the centipede Strigamia maritima and the spider P. tepidariorum[20, 48, 49]. Expression in the ocular region is conserved between E. kanangrensis and these arthropods. However, comparable expression in an early gap-gene-like domain, at the posterior end of the embryo, and in the frontal appendages of E. kanangrensis is not found in these arthropods.
Expression of Wnt4 has been studied in G. marginata, S. maritima and P. tepidariorum[20, 48, 49]. The expression profiles of Wnt4 in these arthropods are completely different. Expression in myriapods is observed throughout the developing embryo, whereas in P. tepidariorum, expression is restricted to the SAZ. None of the expression patterns of Wnt4 in myriapods and E. kanangrensis is the same. Reconstruction of the ancestral arthropod expression pattern of Wnt4 is therefore impossible based on the available data.
Expression of Wnt5 has been investigated in a number of arthropods, including D. melanogaster[50, 51] and T. castaneum, the spiders C. salei[12, 53] and P. tepidariorum, and the myriapods G. marginata and S. maritima. Wnt5 expression in the two spiders is virtually identical, and expression in the other arthropods is in many aspects comparable to that observed in these spiders: Wnt5 is expressed in the ventral nervous system, including the brain, transverse segmental stripes, and the labrum and the limb primordia (and later in the limbs). Expression in the brain, the limb primordia, and in the form of segmental stripes is conserved in E. kanangrensis. This strongly suggests an ancestral and conserved function for Wnt5 in the development of these tissues. This assumption is supported by the fact that Wnt5 is the only Wnt gene (besides wg/Wnt1) that is present in all hitherto studied arthropods (summarized in ; Figure 2). However, apparently lineage-specific expression of Wnt5 includes the expression observed in the heart of spiders and the early gap-gene-like expression in the onychophoran.
Wnt6 expression has been studied in D. melanogaster, T. castaneum, G. marginata and S. maritima. This work has shown that expression in the limbs and brain, and the segmental expression (probably associated with the central nervous system), is conserved among these arthropods. In E. kanangrensis, expression in the ventral nervous system and the limbs appears to be conserved, and may thus represent part of the ancestral expression pattern of Wnt6.
The embryonic expression of Wnt7 has been studied in D. melanogaster[50, 55], T. castaneum, G. marginata, P. tepidariorum and S. maritima[56, 49]. In both insects, Wnt7 is expressed in a segmentally reiterated pattern; a comparable pattern is seen in S. maritima but not G. marginata. One of the two Wnt7 paralogues of P. tepidariorum is expressed in the SAZ, and this pattern is also seen in G. marginata, but not in the onychophoran, or in T. castaneum. In both myriapods, Wnt7 is expressed in the heart, and in the brain, but expression in the labrum is only seen in G. marginata, and expression in the antennae is only present in S. maritima. In summary, while some aspects of the expression of arthropod Wnt7 genes are conserved, none of these expression domains is observed in E. kanangrensis.
Expression of Wnt8 has been studied in D. melanogaster[57, 58], where it is called WntD, and in T. castaneum, as well as in G. marginata and P. tepidariorum. In D. melanogaster, it is first expressed at both poles of the early blastoderm stage embryo. Later during ontogenesis, it is expressed in the mesectoderm and the ventral neurectoderm. In T. castaneum, Wnt8 is only expressed at the posterior pole in blastoderm stage embryos and in the ventral mesoderm in the SAZ. The early expression at the posterior pole and in the SAZ is also conserved in the spider, and functional studies have shown that Wnt8 is involved in posterior segment addition in both T. castaneum and P. tepidariorum[39, 42]. The fact that Wnt8 is only expressed in the primordia of the ocular region and the mandibular segment of G. marginata was therefore unexpected , and contradicted the idea that Wnt8 could possibly play an ancestral and conserved role in arthropod segmentation .
We did not recover an E. kanangrensis Wnt8 in our surveys. It may be that Wnt8 was missed because it is expressed at a low level or because it is not expressed at all during ontogenesis. The possible lack, or nonexpression during ontogenesis, of onychophoran Wnt8, however, supports the possibility that Wnt8 is not an ancestral component of the segmentation machinery of arthropods and onychophorans.
Expression of Wnt9 has been studied in D. melanogaster, T. castaneum[52, 49] and G. marginata. In T. castaneum and S. maritima, Wnt9 is only expressed in a few cells in the gut. In G. marginata, this gene is transiently expressed in segment polarity gene-like segmental stripes, in the appendages including the labrum, and in the SAZ. Later, it is expressed in a dorsal segmental pattern and in the form of stripes in the dorsal extraembryonic tissue. D. melanogaster Wnt9 is also expressed in a segment polarity-like pattern and in the labrum. In E. kanangrensis, at least, expression in the tips of the appendages and the segment polarity gene-like expression are conserved, and this may indeed represent the ancestral expression profile of Wnt9.
Wnt10 expression has so far only been studied in D. melanogaster, S. maritima and T. castaneum. In D. melanogaster, it is expressed in the mesoderm, the developing gut and the central nervous system. In T. castaneum, it is expressed in the cephalic lobes, the appendages and in transverse segmental stripes anteriorly adjacent to the expression of engrailed (en). This expression is also conserved in S. maritima. Segmental expression and expression in the limbs (discussed later) appears to be conserved between T. castaneum, S. maritima and the onychophoran, and may thus represent part of the ancestral expression pattern of Wnt10. This scenario would mean that the expression profile of D. melanogaster Wnt10, however, is derived.
Wnt11 orthologues have been isolated and their expression investigated in T. castaneum, P. tepidariorum, G. marginata, and S. maritima[56, 49]. In P. tepidariorum, there are two paralogues of Wnt11, but only one, Wnt11-2, is expressed in embryos. The expression profile of Wnt11 is similar in the onychophoran, the spider and the myriapods. In all species, Wnt11 is first expressed in the SAZ at the posterior region of the embryo. Later, expression appears in the tips of all appendages (except for S. maritima). However, segment polarity gene-like stripes of expression are only seen in E. kanangrensis. Only in T. castaneum is Wnt11 expressed in the heart.
Wnt11 is likely to play a conserved role during limb development as represented by the strong expression in the tips of the limbs, and segment addition, as represented by the strong expression in the posterior end of the embryos.
Expression of Wnt16 has been described in P. tepidariorum, S. maritima and G. marginata[14, 20] (in 2004, erroneously described as Wnt7). In both the spider and the myriapods, Wnt16 is expressed in transverse segmental stripes anterior and directly adjacent to the expression of en (somewhat unclear for S. maritima) suggesting that it is involved in segmental boundary formation. Wnt16 is also expressed in the developing brain and in the tips and ventral tissue of the limbs. The arthropod Wnt16-expression profile is conserved in E. kanangrensis. One important difference is, however, that the segmental expression of Wnt16 in E. kanangrensis reaches posteriorly into the domain of en expression.
Expression of WntA has been analyzed in T. castaneum, S. maritima and G. marginata[14, 20] (in 2004, erroneously described as Wnt5), and C. salei. Expression in the mandibulate arthropods is comparable. It is strongly expressed in the developing brain, heart, limbs and central nervous system. In E. kanangrensis, WntA is similarly expressed in the brain (head lobe), the limbs and, at later stages, in the ventral nervous system; expression in the heart, however, is not observed in the onychophoran. Expression of WntA in the spider C. salei differs significantly from that in the other arthropods and the onychophoran. The only possibly conserved pattern of WntA is in the SAZ (apart from that, spider WntA is only expressed in small domains in the spinnerets and the chelicerae).
The onychophoran ‘segment’
Onychophorans represent segmented animals, although some of the key characteristics of the arthropods, such as full adult body segmentation with pronounced segmental indentations are not present. The latter is best interpreted as being primitive . Since segmental indentations are lacking in onychophorans, it is difficult to determine the position of the segmental boundaries in the ectoderm. The best approximation may be given by the expression of segment polarity genes that determine segmental and parasegmental boundaries in arthropods [12, 61].
The segment polarity gene network is conserved in arthropods (for example, [11, 12, 14, 62]) and onychophorans [17, 18]. The parasegmental boundary in arthropods lies at the interface between en and wg expressing cells, and the segmental boundary lies posterior to the expression of en (for example, [12, 63]). This means that the parasegmental boundaries of onychophorans are located in the posterior of the limbs, exactly as in arthropods. Determination of the segmental boundaries by means of gene expression patterns is not that clear because the domain of en expression is broadened in ventral tissue  and the posterior border of each stripe thus lies posterior to that of hh, which is the direct downstream target of en in arthropods [15, 64]. Therefore, the segmental boundaries lie either posterior to the en/hh domain and thus directly posterior to the limbs, or the segmental boundaries lie posterior to en and thus somewhat shifted towards the posterior (compare with Figure 13).
Wnt genes and limbs
Interestingly, a recent study revealed two separate functions of wg/Wnt1 in limb development in T. castaneum. One function concerns ventral limb patterning; the other concerns the proximodistal growth of the limbs. It is therefore likely that the expression seen in onychophorans is associated with limb growth, rather than with a function in dorsoventral patterning. This would mean that at least this component of dorsoventral patterning evolved in the lineage leading to the arthropods. The evolution of the dual function of Wnt genes during limb development may be reflected by the situation in the millipede G. marginata, where some Wnt genes are expressed strongly in the tips of the limbs, but only weakly along the ventral side of the limbs . This implies that the expression in the tips as seen in the onychophoran may represent the ancestral state and that ventral expression evolved step by step within the arthropod lineage.
Wnt ligands play important roles during animal development. Our study shows that most of the protostome Wnt ligands are present in onychophorans, and that all of those present are expressed in distinct patterns throughout embryogenesis. It is surprising that eight of twelve Wnt genes are expressed in segmental stripes reminiscent of the expression of classical arthropod segment polarity genes, and that their combined expression covers the complete segments. This suggests that Wnt genes may play a more prominent role in segment regionalization than they do in arthropods, where the expression of Wnt genes is mostly restricted to anterior cells abutting the domain of engrailed (en) expression. Early expression of Wnt2, Wnt4 and Wnt5 in gap-gene-like and Hox-gene-like patterns suggests a contributing role of these genes in giving anterior segments their specific identities. Strong expression of most of the onychophoran Wnt genes in the posterior SAZ might be correlated with a role in germ band elongation or segmentation. Thus, it seems likely that Wnt genes are involved in segment formation, segment regionalization and the definition of segment identity in onychophorans. If these assumptions hold true, the role of Wnt genes in onychophoran segmentation would clearly extend their roles in arthropod segmentation.
metropolis-coupled Markov chain Monte Carlo
phosphate-buffered saline with 0.1% Tween-20
polymerase chain reaction
segment addition zone.
This work has been supported by the Swedish Research Council (grant to GEB) and the Volkswagen Foundation (grant to NP).
We 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. The authors wish to thank Jean Joss, Rolf Ericsson, Robyn Stutchbury and, especially, Noel Tait, for their help during onychophoran collection.
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