Wnt genes in spiders and a harvestman
We reanalyzed the Wnt gene repertoire of Parasteatoda and surveyed the repertoires of these genes in additional spiders, Pholcus and Acanthoscurria, as well as the harvestman Phalangium screening embryonic transcriptomes of all species and the genome of Parasteatoda. Our phylogenetic analysis was similar to those by Harper et al. [21].
The common ancestor of chelicerates likely possessed a “complete” set of the 12 Wnt genes typical for protostomes, despite common lineage-specific losses within this subphylum (Figs. 2, 3; Additional file 1: Fig. S1) [21, 39]. Spiders appear to have lost their Wnt9 and Wnt10 orthologs, while these genes are retained in other chelicerates such as the harvestman Phalangium [21]. Two paralogs of Wnt1, Wnt7, and Wnt11 have been retained (after the WGD in Arachnopulmonata) but the second paralog of Wnt1 has been lost in most true spiders [21]. The lack of a second Wnt4 paralog in Pholcus and the presence of two paralogs of Wnt4 in Acanthoscurria, as well as some lineages of entelegyne spiders [21] suggest that loss of a second Wnt4 gene occurred independently in at least two lineages of spiders (towards Parasteatoda, and towards Pholcus) (Figs. 2, 3). The apparent loss of a second Wnt4 gene in Pholcus may be representative for Haplogynae as a whole as we could not identify a second copy in the published genome of another basally branching haplogyne spider, the recluse Loxosceles reclusa (data not shown). Please note that the lack/loss of a gene is difficult to prove, even in the era of full genome sequencing. Most genomes, although “sequenced” are not complete, or have not been assembled completely. The situation in spiders is even more complicated because of the many duplicated genes and often enlarged intronic regions. Many of the published spider genomes are thus far from having the complete set of genes. The usage of transcriptomic data (as used in our study), using a combination of sequencing methods as well as several rounds of reannotations helps to improve these issues. That is why the Parasteatoda genome is still one of the best annotated genomes present.
Wnt1
In all investigated species, at least one paralog of Wnt1 is expressed in a subset of cells in the pre-cheliceral region, along the ventral side of the appendages (including the opisthosomal limb buds that correspond to the breathing organs and the spinnerets), dorsally in the labrum (except for the harvestman), and in the posterior of the developing embryo (Figs. 4, 5; Additional files 2, 3, 5, 6, 7: Figures S2, S3, S5–7). The posterior expression is either corresponding to the hindgut primordium that is located posterior to the segment-addition zone (marked with SAZ), or the posterior part of the SAZ. While this expression appears early during development in other spiders suggesting a role as posterior patterning gene, in Parasteatoda this expression is restricted to later developmental stages indicating that it may indeed correspond to the hindgut rather than be involved in segment addition (Fig. 4B, C). Interestingly, there are two paralogs of Wnt1 in Acanthoscurria. The second paralog, Wnt1.2, is exclusively expressed in the SAZ (Fig. 4M, N), while the other paralog, Wnt1, is expressed similar to the single Wnt1 gene in the other species, but is lacking expression in the SAZ (Fig. 4I–L). This represents an impressive example of sub-functionalization after WGD. With the exception of Acanthoscurria, for all species studied dorsal stripes of expression appear in the opisthosoma late during embryogenesis (Figs. 4, 5). Only in Parasteatoda, there is a line of expression dorsal in the head and the limb-bearing segments (Fig. 4C, D).
In true spiders, Wnt1 is expressed in the form of segment polarity gene (SPG)-like transverse stripes, but such stripes are restricted to some of the head segments [39] (Fig. 4F). In Acanthoscurria, there are no SPG-like stripes of expression (Fig. 4I–N). In Phalangium, however, SPG-like stripes are present early during development, and in all developing segments (including posteriorly added segments) (Fig. 5A–F). Note that expression of Wnt1 in the developing books lungs of Parasteatoda is in the form of three separate domains as previously described for another entelegyne spider, Cupiennius salei [9] (Fig. 4C(inlay)). Expression patterns of spider and harvestman Wnt1 genes are summarized in schematic Figs. 4O and 5G, respectively.
Wnt2
We identified a single Wnt2 ortholog in all spider species, but not in the harvestman. In all spiders, Wnt2 is expressed in a subset of cells in the pre-cheliceral region (Fig. 6; Additional file 3, 6, 7: Fig. S3B, S6B, S7B). Notably, this domain appears already during early germ band stages in Parasteatoda and covers a larger area of the brain in later stages compared to Pholcus and Acanthoscurria, the latter displaying the smallest brain expression domain (Fig. 6). In Pholcus and Acanthoscurria, Wnt2 is expressed in the SAZ throughout development, but in Parasteatoda, there is no such posterior expression (Fig. 6). Similarly, in Pholcus and Acanthoscurria Wnt2 is expressed along the ventral side of the prosomal appendages (except for the labrum), but in Parasteatoda expression is restricted to some dot-like domains along the ventral side of the appendages (Fig. 6; Additional files 3, 6, 7: Figs. S3, S6, S7). Expression of spider Wnt2 genes is summarized in the schematic Fig. 6K.
Wnt4
In most spiders, there are two paralogs of Wnt4 [21], in Pholcus and Parasteatoda, however, only one Wnt4 is present (Parasteatoda) or has been identified in an embryonic transcriptome (Pholcus) (Fig. 3). Only in Acanthoscurria, we were able to identify two paralogs of Wnt4.
Wnt4 exhibits quite diverse expression among spiders and between these animals and the harvestman (Fig. 7; Additional files 3, 5, 6, 7: Fig. S3, S5–S7). The only common features are the dot-like domains in the distal ectoderm of the legs and pedipalps of spiders, and the expression in the labrum (except for Acanthoscurria). In the harvestman, however, expression in the pedipalps and legs is different to the spiders and restricted to a distal portion of the limb mesoderm (cf. Additional files 3, 5, 6, 7: Fig. S3, S5–S7). Although expression in the legs and pedipalps of spiders is mainly restricted to ventral tissue, one of the two tarantula Wnt4 genes (Wnt4.2) is expressed in dorsal (and rather proximal) domains (cf. panels C and D of Additional file 3: Figure S3). Patterns of presence and absence in the prosomal appendages of spiders differs between the investigated species (Additional files 3, 5, 6, 7: Figs. S3, S5–S7). In all species (except Acanthoscurria), there is a complex pattern of expression in the pre-cheliceral region (Fig. 7). In all species, Wnt4 is expressed at the posterior pole of the developing embryo, although the signal in Acanthoscurria is very weak and thus may represent background (Fig. 7). In true spiders, expression in the posterior is clear, but only appears at relatively late developmental stages, while comparative expression appears very early during germ band formation in the harvestman (Fig. 7O). Only in the tarantula, one of the two Wnt4 paralogs (Wnt4.1) is expressed in SPG-like stripes early during development (Fig. 7H), and in the harvestman a unique ventral expression appears during later stages in the opisthosoma (Fig. 7Q). Another unique expression is present for Parasteatoda Wnt4 forming a dorsal stripe separating the prosoma from the opisthosoma (Fig. 7C). The expression patterns of Wnt4 genes are too diverse to identify possible patterns of sub- or neo-functionalization in Acanthoscurria. Here, expression patterns of other chelicerate species that retained two paralogs could help to clarify an ancestral feature of Wnt4. Expression patterns of Wnt4 genes are summarized in the schematic Fig. 7S.
Wnt5
In Parasteatoda and Pholcus, expression of Wnt5 starts after germ band formation and shortly before the limb buds begin to grow out (Fig. 8A, E). The same pattern is seen in the early germ bands of Acanthoscurria and Phalangium, but we do not know if expression starts already earlier in these species (Fig. 8I, N). This expression most likely correlates with the limb primordia. Furthermore, in all species, Wnt5 is expressed in a large domain of the pre-cheliceral region and the ventral nervous system (Fig. 8; Additional files 5, 7: Figs. S5C, S7D). Wnt5 is also expressed in all appendages, including the opisthosomal limb buds, but not in the labrum (with the exception of dot-like domains late in Acanthoscurria and Pholcus) (Fig. 8; Additional files 3, 5, 7: Figs. S3, S5–S7). Interestingly, in all species, the limb expression resembles leg-gap gene like domains. In all species, Wnt5 is also expressed in the dorsum of the opisthosomal segments; likely, this expression is correlated with the development of the heart (arrowhead in Figs. 8D, G, H, M, O–Q, S) (cf. [37]). In the three spiders, but not in the harvestman, Wnt5 is also expressed is in the stomodeum (Fig. 8C, F, J; Additional file 7: Figure S7D). Wnt5 expression is summarized in the schematic Fig. 8T.
Wnt6
In all species, Wnt6 is expressed along the ventral side of all appendages, including the opisthosomal limb buds (Fig. 9; Additional files 3, 5, 6, 7: Figs. S3, S5–7). In the labrum, Wnt6 is expressed dorsally but note that Phalangium Wnt6 is not expressed in the labrum at all (Fig. 9H, L; Additional files 5, 7: Figs. S5D, S7E). In Parasteatoda, expression starts when the limb buds begin to grow out (Fig. 9A). In Acanthoscurria, the earliest Wnt6 expression commences just before the formation of the limb buds in a SPG-like fashion (Fig. 9J). In Pholcus and Phalangium, expression starts earlier and in SPG-like transverse stripes before the onset of limb bud development (Fig. 9E, N). The anterior-most stripe is correlated with later expression in the pre-cheliceral region. This expression was not observed in Parasteatoda or Acanthoscurria (Fig. 9). The early stripes later become restricted to expression in the developing appendages and thin stripes of expression ventral to the base of the appendages, most prominently seen in Phalangium where the germ band halves do not split unlike in spiders (Fig. 9R). In Phalangium and Parasteatoda, Wnt6 is expressed in the SAZ, but while this expression is already present in early stages of the harvestman, expression in this spider appears later during germ band extension (Figs. 9A, N). The other two spiders do not express Wnt6 posteriorly (Fig. 9), except for an early transient posterior domain in Pholcus (Fig. 9E). In all spiders, Wnt6 is also expressed dorsal to the base of the appendages, which is especially prominent in the opisthosoma (Fig. 9). This expression is likely correlated with the development of the heart and in Acanthoscurria, the developing heart tube itself expresses Wnt6 (Fig. 9K, M). Additional expression of Wnt6 was observed in the stomodaeum of the harvestman (Fig. 9S), and in the form of transverse segmental stripes in the ventral sulcus (the region between the split germ band halves) of the tarantula (Fig. 9L). Similar stripes of expression in the ventral sulcus have been reported for netrin expression in spiders including Parasteatoda, suggesting that Wnt6 may be involved in axonal guidance [48]. Expression of Wnt6 is summarized in the schematic Fig. 9T.
Wnt7
All spiders investigated here possess two Wnt7 paralogs (Fig. 3). In true spiders, one Wnt7 gene (Wnt7.1) is expressed in the posterior SAZ region (Fig. 10A, B, D, H, I). While this is the only expression of Wnt7.1 observed in Pholcus, Parasteatoda Wnt7.1 is also expressed in the developing limb buds including the opisthosomal buds, and in part of the brain and the ventral nervous system (Fig. 10B–D). In the limbs, this expression is predominantly present along the ventral side, but a dot of expression is also visible proximally and dorsal (Additional file 7: Fig. S7F). In the tarantula, Wnt7.1 expression is restricted to late embryonic stages and mainly in the ventral ectoderm of the appendages, except for the labrum that does not express Wnt7.1 (Fig. 11A, B; Additional file 4: Figure S4A).
In all spiders, Wnt7.2 is expressed in the appendages (Figs. 10, 11; Additional files 4, 6, 7: Figs. S4B, S6F, S7G). In Parasteatoda, Wnt7.2 is expressed in the form of several dot-like domains along the dorsum of the labrum, the pedipalps, the legs and the opisthosomal limb buds, but ventral in the chelicerae (Additional file 7: Figure S7G). In addition, there is a dot-like expression ventrally and close to the tip of the legs. In Pholcus, however, expression in chelicerae, pedipalps, legs, and opisthosomal appendages is restricted to the dorsal-proximal region (Additional file 6: Figure S6F). In Acanthoscurria, expression in the chelicerae is ventral, as described for Parasteatoda, and expression in the pedipalps and legs is restricted to a dorsal-proximal patch as described for Pholcus (Additional file 4: Figure S4B). Additionally, Wnt7.2 is expressed in four dominant large domains in the pre-cheliceral region of Parasteatoda (Fig. 10E; Additional file 7: Figure S7G). Similar expression is present in Pholcus and Acanthoscurria albeit in smaller domains (Figs. 10N, 11C). In the spiders Parasteatoda and Pholcus, Wnt7.1 and Wnt7.2, respectively, are also expressed in the developing ventral nervous system (Fig. 10D, M). In the harvestman Phalangium, the single copy of Wnt7 is only expressed in the dorsal-proximal region of the pedipalps and the legs (but not the labrum or the chelicerae) (Fig. 11F–H; Additional file 5: Figure S5E). Expression of Wnt7 genes is summarized in schematic Figs. 10O, 11I.
Wnt8
In all investigated spiders, Wnt8 is expressed in the ventral ectoderm of the chelicerae, the pedipalps, the legs and the opisthosomal limb buds (Additional files 4, 6, 7: Figs. S4C, S6G, S7H) but only in Parasteatoda expression is also present dorsally in the labrum (Additional file 7: Fig. S7H). In Pholcus and Acanthoscurria (but not Parasteatoda), Wnt8 is expressed in the stomodaeum (Fig. 12F(inlay), J). In all spiders, expression starts early during embryogenesis in the form of transverse segmental stripes that are reminiscent of SPG expression (Fig. 12; Additional file 8: Figure S8). In Parasteatoda, expression starts already during the germ disc stage as a central patch and a ring close to the rim of the disc (Fig. 12A). The latter transforms into expression in the pre-cheliceral region, which is also present in the other spiders. The central patch of expression in Parasteatoda, however, that later represents expression in the SAZ, is not present in Pholcus. Indeed, the earlier reported strong expression of Wnt8 in the SAZ of Parasteatoda [58] is neither present in the entelegyne spider Pholcus nor the tarantula Acanthoscurria. Like a typical SPG, in all spiders Wnt8 is expressed in the form of transverse stripes in all newly forming posterior segments (Fig. 12C–F, K; Additional file 8: Fig. 8D). In Phalangium, Wnt8 expression is restricted to two domains in the pre-cheliceral region (Fig. 12M, N). Expression of Wnt8 is summarized in Fig. 12O.
Wnt9 and Wnt10
We did not identify any orthologs of Wnt9 and Wnt10 in the spider species studied here. In Phalangium, however, we found representatives of both subfamilies (Fig. 3). Wnt9 is first expressed in a SPG-like pattern as transverse segmental stripes covering the region where the limbs will form and the most ventral tissue of the embryo (Fig. 13A). These early stripes correspond to a domain in the anterior head, the chelicerae-bearing segment, the pedipalpal segment and the first leg-bearing segments. Additional expression is present in the very posterior of the embryo (likely the hindgut primordium) and when posterior segments are added, Wnt9 is expressed in similar transverse stripes in these segments (Fig. 13B–D). As the appendages develop, expression is restricted to a central sector along the ventral side of the chelicerae, the pedipalps and the legs (and their endites), but in the labrum Wnt9 is dorsally expressed (Fig. 13B–D; Additional file 5: Fig. S5F). Later during development, expression appears in the stomodaeum (Fig. 13C).
Expression of Wnt10 also starts early during development and in the form of transverse stripes; note however, that these stripes are not continuous (cf. expression of Wnt9). Instead, expression in the ventral region of the embryo is missing (Fig. 13F–I). We assume that these stripes are correlated with the primordia of the appendages. The most anterior expression domains are located in the pre-cheliceral region. Later during development, expression is observed centrally along the ventral side of the appendages (including the endites) (Fig. 13G–I; Additional file 5: Figure S5G). Unlike Wnt9, Wnt10 is not expressed in the labrum. Expression in the posterior pole of the embryo is comparable to that of Wnt9, but no stripes were observed in the opisthosomal segments (Fig. 13H, I). Late during embryogenesis, expression of Wnt10 appears in the stomodaeum (Additional file 5: Figure S5G). Expression of Wnt9 and Wnt10 is summarized in Fig. 13E, J, respectively.
Wnt11
In Parasteatoda and Pholcus, Wnt11 is represented by two paralogs (Fig. 3). However, in both species, expression of Wnt11.1 was not detected in any of the investigated embryonic stages (cf. [39]. In Acanthoscurria and Phalangium only one copy of Wnt11 was found. In Parasteatoda and Phalangium, expression of Wnt11.2 and Wnt11, respectively, appears early during embryogenesis in the SAZ (Fig. 14A, B, J), but in Pholcus and Acanthoscurria, there is no such posterior expression (Fig. 14F, G, I). In the appendages of all investigated animals (including the opisthosomal buds), expression was observed in the ventral ectoderm, except for the labrum where expression is dorsal (the labrum of the tarantula and the harvestman do not express Wnt11) (Fig. 14; Additional files 4, 5, 6, 7: Figs. S4D, S5H, S6H, S7I). Expression in the chelicerae of the harvestman is internal, likely mesodermal (Additional file 5: Fig. S5H). Expression of Wnt11 is summarized in Fig. 14M.
Wnt16
In all investigated species, Wnt16 is expressed in a SPG-like pattern in the form of transverse segmental stripes (Fig. 15; Additional file 9: Fig. S9). In Pholcus, Acanthoscurria and Phalangium, these stripes appear early during development (cf. Fig. 15E, I, N, O with Additional file 9: Figure S9B), while in Parasteatoda the expression starts later coinciding with limb bud formation (Fig. 15A). In spiders, there is no (or only weak) expression in the posterior SAZ, but in the harvestman, Wnt16 is dominantly expressed in the SAZ (Fig. 15O, Q). In all species, Wnt16 is also expressed in the pre-cheliceral region and the stomodaeum (Fig. 15A, B, E–G, J, L, P, R, S; Additional files 5, 7: Figs. S5I, S7J). In Acanthoscurria, Wnt16 is expressed on the dorsal side of the labrum and two thin longitudinal stripes of expression run on either side of the stomodaeum (Fig. 15L). Common to all analyzed species, expression in the appendages is restricted to the ventral side including the ventral sector of the endites (if present); in the labrum, expression is always dorsal (Fig. 15; Additional files 4, 5, 6, 7: Figs. S4E, S5I, S6I, S7J). In all spiders, Wnt16 is expressed in the form of short stripes (or patches) dorsal to the opisthosomal limb buds (Fig. 15D, H, M). Comparable expression is also present in the opisthosomal and the leg-bearing segments in Phalangium (Fig. 15S). Expression of Wnt16 is summarized in Fig. 15T.
WntA
In all species, WntA is expressed in the SAZ (Fig. 16). In all species, except Parasteatoda, expression is present in the pre-cheliceral region (Fig. 16E, F, K, O, S), and the ventral nervous system along either side of the midline (Fig. 16E–H, L, M, R). Expression in the developing appendages is diverse. In Phalangium, expression of WntA in chelicerae, pedipalps and legs is exclusively mesodermal.
In Parasteatoda and Pholcus WntA is expressed in one or several patches in the dorsal ectoderm of the legs, pedipalps and the chelicerae (Additional files 6, 7: Figs. S6J, S7K). Additionally, WntA is expressed in the mesoderm of these appendages in Pholcus (Additional file 6: Figure S6J). In Acanthoscurria expression in the limbs is weak, but still dorsal and distal ectodermal expression domains as well as expression in the mesoderm are present in at least the pedipalps and the legs (Additional file 4: Figure S4F). Only in Parasteatoda, WntA expression was also observed in the dorsal tissue of the labrum (Fig. 16C; Additional file 7: Figure S7K). Expression of WntA is summarized in Fig. 16T.