Open Access

Duplicated Hox genes in the spider Cupiennius salei

  • Evelyn E Schwager1,
  • Michael Schoppmeier1, 2,
  • Matthias Pechmann1 and
  • Wim GM Damen1Email author
Frontiers in Zoology20074:10

https://doi.org/10.1186/1742-9994-4-10

Received: 14 December 2006

Accepted: 13 March 2007

Published: 13 March 2007

Abstract

Background

Hox genes are expressed in specific domains along the anterior posterior body axis and define the regional identity. In most animals these genes are organized in a single cluster in the genome and the order of the genes in the cluster is correlated with the anterior to posterior expression of the genes in the embryo. The conserved order of the various Hox gene orthologs in the cluster among most bilaterians implies that such a Hox cluster was present in their last common ancestor. Vertebrates are the only metazoans so far that have been shown to contain duplicated Hox clusters, while all other bilaterians seem to possess only a single cluster.

Results

We here show that at least three Hox genes of the spider Cupiennius salei are present as two copies in this spider. In addition to the previously described duplicated Ultrabithorax gene, we here present sequence and expression data of a second Deformed gene, and of two Sex comb reduced genes. In addition, we describe the sequence and expression of the Cupiennius proboscipedia gene. The spider Cupiennius salei is the first chelicerate for which orthologs of all ten classes of arthropod Hox genes have been described. The posterior expression boundary of all anterior Hox genes is at the tagma border of the prosoma and opisthosoma, while the posterior boundary of the posterior Hox genes is at the posterior end of the embryo.

Conclusion

The presence of at least three duplicated Hox genes points to a major duplication event in the lineage to this spider, perhaps even of the complete Hox cluster as has taken place in the lineage to the vertebrates. The combined data of all Cupiennius Hox genes reveal the existence of two distinct posterior expression boundaries that correspond to morphological tagmata boundaries.

Background

Hox genes are found in all metazoan phyla. They are active in distinct domains along the main body axis and direct the morphogenesis of segment-specific structures via the activation of downstream target genes. Hox genes are important factors in the evolution of animal body plans. They share three key traits [1]: (1) they are basically organized in a cluster in the genome, (2) there is a correlation between the 3'-5' order of the genes in the genome and the anterior to posterior order of expression of the genes, and (3) the protein encoded by each of the genes contains a homeobox, a highly conserved 60 amino acid sequence that is a DNA binding motif [2].

The Hox genes primarily are involved in providing the embryo with positional information. This is most obvious from experiments with mutants that lack a particular Hox gene or from embryos in which a particular Hox gene is misexpressed. Such embryos produce structures at the incorrect position, as the affected cells seem to misunderstand their location within the embryo. For instance when a particular Hox gene is absent or is misexpressed in the fruit fly Drosophila melanogaster, the affected segments get the identity of another segment [e.g. [3, 4]]. Famous examples are the four-winged Drosophila fly, in which the halteres on the third thoracic segments are transformed to wings, or the flies with legs at the position of the antennae. This is homeosis, and the mutations are homeotic transformations. The Hox genes thus act as selector genes that select one anterior-posterior identity over another along the main body axis in the embryo, while their downstream target genes actually act as realizator genes that make the structure specific for each location [summarized in [5, 6]].

Due to the widespread sampling of Hox genes from a large variety of metazoans, the evolution of Hox genes is well characterized. Gene duplications played an important role in the evolution of the Hox genes. Recent data on cnidarians [7] suggest that the last common ancestor of the cnidarians and bilaterians had a Hox cluster consisting of two anterior genes (a Hox1/2 and a Hox3 gene), and that the Hox cluster subsequently expanded via internal duplications in the lineages leading to the cnidarians and the bilaterians. The last common ancestor of the bilaterians (animals with a bilateral symmetry) presumably still contained such a cluster of three genes as seen in today's acoel flatworms, which may represent the closest approximation of the ancestral bilaterian [8]. The last common ancestor of the other bilaterians (the protosome/deuterostome ancestor) at least contained seven different Hox genes, maybe even nine or more [9], implying several Hox gene duplication events in this lineage after the divergence of the acoel flatworms [8]. The different genes in the Hox complex are most likely the result of tandem duplications followed by sequence divergence [9, 10]. In vertebrates the complete Hox cluster has been duplicated twice, presumably via whole genome duplications, resulting in four clusters in tetrapods, while in teleost fish additional duplication events took place [11].

Arthropod Hox genes can basically be assigned to ten different classes and seem to be present in a single Hox cluster [12]. In the chelicerates (spiders, scorpions, mites, ticks, horseshoe crabs) however there are examples of duplicated Hox genes. In a PCR survey Cartwright et al. [13] found 28 different small homeobox fragments of Hox genes in the horseshoe crab Limulus polyphemus. They could identify one to four representatives for each Hox gene class suggesting the presence of multiple Hox clusters in an invertebrate. Additional data for duplications of Hox genes come from two spiders, Achaearanea tepidariorum and Cupiennius salei. Two copies of the Deformed (Dfd) gene have been described for Achaearanea [14], and a duplicated Ultrabithorax (Ubx) gene has been described for Cupiennius [15]. In addition to these chelicerates there is one example of a duplicated Hox gene in a myriapod; a duplicated Dfd gene has been described for the geophilomorph centipede Pachymerium ferrugineum [16].

In the present paper we describe four new Hox genes from the spider Cupiennius salei: a proboscipedia gene (Cs-pb), a second Dfd gene (Cs-Dfd-2), and two Sex comb reduced (Cs-Scr) genes. Our data shows that at least three Hox genes (Dfd, Scr, and Ubx) are duplicated in the spider C. salei. Furthermore, pb and Scr orthologs have not been described from C. salei before. In previous work we described the sequence and expression of orthologs of eight classes of Hox genes from the spider C. salei: labial (Cs-lab), Hox3 (Cs-Hox3), Deformed (Cs-Dfd-1), fushi tarazu (ftz), Antennapedia (Cs-Antp), Ultrabithorax (Cs-Ubx-1 and Cs-Ubx-2), abdominal-A (Cs-abdA), and Abdominal-B (Cs-AbdB) [15, 1719]. With our new data on pb and Scr, the Central American wander spider Cupiennius salei becomes the first chelicerate for which orthologs of all ten arthropod Hox genes have been described.

Results

Spider Hox genes

We isolated fragments of proboscipedia (Cs-pb), two copies of Sex comb reduced (Cs-Scr-1 and Cs-Scr-2), and a second copy of Deformed (Cs-Dfd-2) from the spider Cupiennius salei. Alignment with chelicerate and other arthropod sequences unambiguously show that these are Cupiennius orthologs of these Hox genes (Fig. 1). pb and Scr class Hox genes have not been recovered in Cupiennius before, but some data are available from other chelicerates: the mite Archegozetes longisetosus, the common house spider Achaearanea tepidariorum and the seaspider Endeis spinosa [14, 19, 21]. However, with the isolation of pb and Scr from Cupiennius, this spider is the first chelicerate species for which orthologs of all ten arthropod Hox gene classes are described.
Figure 1

Alignment of chelicerate Proboscipedia, Deformed, Sex comb reduced, Ultrabithorax sequences. Aligned is a fragment covering the hexapeptide (core in red) and homeodomain (blue) plus some flanking sequences. The deduced proteins sequences are from the chelicerates Cupiennius salei (Cs; American wandering spider), Achaearanea tepidariorum (At; common house spider), Archegozetes longisetosus (Al; mite), Endeis spinosa (Es; seaspider) the fruit fly Drosophila melanogaster (Dm; insect), the centipedes Pachymerium ferrugineum (Pf; myriapod), Strigamia maritima (Sm; myriapod), and the isopod Porcellio scaber (Ps; crustacean). Amino acids identical to the upper sequence are indicated as dashed "-"; dots "." are used to indicate gaps that have been introduced for alignment purposes. The linker between hexapeptide and homeodomain in the Es-pb sequence is rather long, therefore 19aa are not shown (marked as //19aa// in the alignment). Please note, some of the At, Al and Pf sequences from the GenBank still contain ambiguous sites that result in an unknown amino acid in the deduced protein sequence; these are indicated as "X" in the alignment. Accession numbers of the sequences used: Cs: AM419029, CAA07498, AM419032, AM419030, AM419031, CAA07500, CAA07501; At: AF151997, AF151995, AF151996, AF151998; Al: AF071406, AF071403, AF071407; Es: ABD46724. ABD46727, ABD46728; Dm: CAA45272, NP_477201, NP_524248, NP_536752; Pf: CAB75743, CAB75746; Sm: ABD16212, Ps: BAE97002.

More importantly, it becomes clear that at least three Hox genes in Cupiennius are present as duplicate copies. There are two Dfd orthologs [[15], this paper], two Scr orthologs (this paper), and two Ubx orthologs [15]. Despite the similarities in the amino acid sequence and especially within the homeodomain, the two copies are significantly different from each other on the DNA sequence level (not shown) and thus are different genes and not different alleles. At the moment it is unclear whether there are additional copies of other Cupiennius Hox genes. Abzhanov et al [14] also described two Dfd genes for another spider (Achaearanea tepidariorum), however, for one of them they only obtained a small PCR fragment encoding 27 amino acids within the highly conserved homeodomain. We recovered additional sequence information of this Achaearanea Dfd gene (At-Dfd-1) via RACE-PCR (Fig 1). The Cs-Dfd-1 [15] and At-Dfd-1 sequences are more similar to each other than to the newly identified Cs-Dfd-2 sequence or to the At-Dfd-2 [14] sequence. Also Cs-Dfd-2 and At-Dfd-2 are more similar to each other than to Cs-Dfd-1 or At-Dfd-1. This is most obvious from the sequence between hexapeptide and homeodomain. Based on the sequences (Fig. 1) we propose that Cs-Dfd-1 and At-Dfd-1 are gene orthologs and that Cs-Dfd-2 and At-Dfd-2 are gene orthologs. Also the expression patterns of At-Dfd-1 [14] and Cs-Dfd-1 [15] in the legs are remarkably similar (see also below). Therefore, the duplication of Dfd presumably was already present in the last common ancestor of these two spiders.

There is another arthropod, the geophilomorph centipede Pachymerium ferrugineum (Myriapoda), that contains two copies of the Dfd gene [16]. However, these two centipede Dfd genes are more similar to each other than to any of the spider genes (Fig. 1). In addition, no Hox gene duplications have been described for other myriapods, e.g. the centipede Lithobius atkinsoni [22] and the millipede Glomeris marginata [23]. This suggests that the two Dfd genes in Pachymerium are the result of an independent duplication event in the geophilomorph centipedes.

Expression patterns of Cs-pb, Cs-Dfd and Cs-Scr

The expression of the Hox genes was studied via in situ hybridizations. Cs-pb is expressed in the pedipalpal segment and the four walking leg segments (L1-L4) (Fig. 2). This is similar to the common house spider Achaearanea and the mite Archegozetes longisetosus [14, 20]. Cs-pb thus is expressed in the same segments as Cs-lab and Cs-Hox3 [15, 17] (see also summary in Figure 6). At the limb bud stage (Fig. 2A) the expression is most obvious in the appendages (pedipalps and walking legs) but there is also some expression in the ventral ectoderm. At the inversion stage (Fig. 2B) Cs-pb expression is also clearly visible in the ventral ectoderm. We never observed any expression in the cheliceral segment or the opisthosomal segments.
Figure 2

Expression pattern of the Cs-pb gene. (A) Limb bud stage, proboscipedia (pb) is expressed in the pedipalpal segment (Pp) and walking leg segments (L1-L4). The expression is most prominent in the appendages itself, but there is also some weaker expression in the ventral ectoderm (arrows). There is no expression in the cheliceral segment (Ch) or in the opisthosomal segments. (B) Inversion stage, pb is expressed in the appendages of the Pp and L1-L4 segments and in spots (arrowheads) in the ectoderm ventral to the appendages. Also at these older stages the cheliceral and opisthosomal segments are free of expression. Both embryos: ventral view.

Expression of the Cs-Dfd-2 gene is limited to the four segments that bear the walking legs (L1-L4) (Fig. 3D–F). These are the same segments that express the Cs-Dfd-1 gene (Fig. 3A–C). However, there are differences in the intrasegmental domains of the expression of the two Cs-Dfd paralogs. Neither are homogenously expressed, but each gene is expressed in a distinct pattern within the leg segments. Most prominent is the very strong expression of Cs-Dfd-1 at the most distal tip of the legs. Although Cs-Dfd-2 also is expressed in the distal tip, this expression is not as prominent as the one of Cs-Dfd-1. Furthermore, while Cs-Dfd-1 is expressed in all four walking legs at the same intensity (Fig. 3C), expression of Cs-Dfd-2 is weaker in L3 and L4 compared to L1 and L2 (Fig. 3F). Another difference is the strength of expression in the ectoderm ventral to the legs: Cs-Dfd-2 is only weakly expressed, while Cs-Dfd-1 is strongly expressed here (compare Fig 3A and Fig 3D). The common house spider Achaearanea tepidariorum also contains two Dfd genes [14]. Comparison of the expression pattern of the two Cupiennius Dfd genes with the two Achaearanea Dfd genes [14] shows that Cs-Dfd-1 and At-Dfd-1 show similarities in their expression patterns. Most typical is the strong expression at the distal tip of the leg, which is much less prominent for Cs-Dfd-2 and At-Dfd-2. This prominent expression in the tip of the leg of Cs-Dfd-1 is most obvious when the colour reaction of the in situ develops (not shown).
Figure 3

Expression pattern of the Cs-Dfd-1 and Cs-Dfd-2 genes. The Cs-Dfd-1 gene (A-C) and the Cs-Dfd-2 gene (D-F) are expressed in the four segments that bear the walking legs (L1-L4). Expression is seen in the appendages as well as in the ventral ectoderm. The ventral ectoderm expression of Cs-Dfd-1 is stronger than the one of Cs-Dfd-2, especially at the older stages (B and C compared with E and F). The expression patterns in the legs are different for the two genes (most obvious in C an F), while in addition, Cs-Dfd-2 expression in L3 and L4 is weaker compared to the expression in L1 and L2. The level of Cs-Dfd-1 expression is similar for all four legs. A and D: ventral view of limb bud stage, B and E: ventral view of limb stage before inversion, C and F: lateral view of inversion stage. There is never ever expression of any of the two spider Dfd genes in the cheliceral (Ch), pedipalpal (Pp), or opisthosomal segments.

Cs-Scr-1 and Cs-Scr- 2 also are expressed in similar but not identical patterns. Cs -Scr-1 is initially expressed in the second, third and fourth walking leg segment (L2-L4) (Fig 4A). In the appendages, expression first appears only in the walking legs of L3 and L4 (Fig. 4B) and only later, but weaker, in the walking legs of L2 (Fig 4C). Cs-Scr-2 is also initially expressed in L2-L4, but the expression is not as widespread as the Cs-Scr-1 expression is as it is restricted to some small spots in the ventral ectoderm (Fig. 4D). Later expression is seen in the legs of L3 and L4 (Fig. 4E) but we never observed expression of Cs-Scr-2 in the legs of L2 (Fig. 4F). As with the two Dfd genes the patterns within the legs also differ for the two Cupiennius Scr genes (compare Fig. 4C and Fig. 4F).
Figure 4

Expression pattern of the Cs-Scr-1 and Cs-Scr-2 genes. Expression of the Cs-Scr-1 gene (A-C) and the Cs-Scr-2 gene (D-F). At the limb bud stage Scr-1 is more or less homogenously expressed in L2, L3 and L4 (arrow in A). At later stages there is expression in rings in the legs of L3 and L4 (B) and later also weak expression in the legs of L2 (C). Scr-2 expression starts as small spots in the ventral ectoderm of L2, L3 and L4 (arrowheads in D) that persist during further development (E,F). Later there is also Scr-2 expression in the legs of L3 and L4, but in contrast to Scr1 we never observed expression in the legs of L2 (F). Ch: cheliceres, Pp: pedipalps, L1-L4: walking leg 1–4.

The expression patterns for Cs-Ubx-1 and Cs-Ubx-2 have been described before [15]. The anterior border of Cs-Ubx-1 is slightly more anterior than that of Cs-Ubx-2 [15]. There are small intrasegmental expression differences between the two Ubx genes. Cs-Ubx-2 is more homogeneously expressed compared to Cs-Ubx-1 (Fig 5A,B).
Figure 5

Expression pattern of the Cs-Ubx-1 and Cs-Ubx-2 genes. The anterior expression border of both Ubx genes is in the second opisthosomal segment (O2). The one of Cs-Ubx-1 (A) is at the anterior edge of O2, while the one of Cs-Ubx-2 is at posterior portion of O2 and corresponds to the parasegment boundary [15]. The opisthosomal limb primordia that will form the respiratory organs and spinnerets are visible on O2-O5. Abbreviations: L4: walking leg 4, O1-O6: opisthosomal segment 1–6.

Figure 6

Schematic representation of the segmental expression patterns of the Hox genes in the spider Cupiennius salei. The segmental organization of the spider and the expression domains of engrailed (en; dark blue) and wingless (wg; bright blue) are shown at the top [see Ref. 31], below are the expression domains of the various Hox genes. Data from: this paper, Refs. [15, 17-19]. Note that the posterior expression domain of the anterior Hox genes (lab, pb, Hox3, Dfd, Scr, ftz) is in the segment of the fourth walking leg and corresponds to the tagma boundary between prosoma and opisthosoma. The expression of the posterior Hox genes (Antp, Ubx, abdA, AbdB) expands to the very posterior end of the embryo. Abbreviations: Oc: ocular; Ch: cheliceral segment; Pp: pedipalpal segment; L1-L4: walking leg segment 1–4; O1-O12: opisthosomal segment 1–12.

Discussion

All ten arthropod Hox genes are present in the spider Cupiennius salei

The combined data from other arthropods [e.g. [8, 19, 20]], summarized in Hughes and Kaufman [12], imply that the Hox complex of the last common ancestor of all arthropods contained ten Hox genes. The present data of the Cs-pb and Cs-Scr genes combined with our previous work [15, 1719] show that at least one copy of each of the ten arthropod Hox genes is present in the spider Cupiennius (Fig. 6). At least three of the Hox genes are even present as two copies (see below). The expression data on Cs-pb and Cs-Scr make Cupiennius the first chelicerate for which expression data are known for all ten different arthropod Hox genes; previously the chelicerate data were an assemblage from three different species [see also Ref. [12]].

At least three duplicated Hox genes in the spider Cupiennius salei

Our data show that at least three Hox genes are present as two copies in Cupiennius [combined data from this paper and Ref. [15]]. There are previous reports on duplication of Hox genes in chelicerates. Cartwright et al. [13] could identify one to four representatives per Hox gene class in the horseshoe crab Limulus polyphemus. However, there is no expression data for the Limulus Hox genes. In the spiders Achaearanea [14] and Cupiennius [15], previous one duplicated Hox gene each had been described. For mite and pycnogonids no duplicated Hox genes have been described [e.g. [20, 21]].

In all three cases in Cupiennius (Dfd, Scr, and Ubx), the two paralogs are expressed in comparable but not identical domains. They are expressed in the same segments with differences in the intrasegmental patterns. This shows a striking similarity with what is seen for the duplicated Hox genes of the various paralogous groups in vertebrates that are also expressed in similar but not completely identical expression domains [24].

Gene duplications offer several possible outcomes [25]. One option is that one of the copies gets silenced or lost again during evolution. A second option is that one copy retains the ancestral function, freeing the other copy to diverge and evolve new functions (neofunctionalization). A third possibility is that each of the two copies performs a different subset of the ancestral functions (subfunctionalization). The differences in the intrasegmental expression patterns of the two copies in Cupiennius suggest that each of the two copies performs different functions that must be the result of either a neofunctionalization or a subfuntionalization event. As there is no genomic information available yet, it remains unclear whether there are additional duplicated Hox genes in Cupiennius (see also next section).

An important question is why duplicated Hox genes are present in the spider and why they are retained? Are they remnants of a large duplication event that are maintained because of neofunctionalization or subfunctionalization events? Or is there another evolutionary advantage for the spider to have multiple copies of some Hox genes? Presently it is difficult to answer these questions. In chelicerates there seems to be a tendency towards having more Hox genes [this paper, [1315]], this in contrast to insects where there is a reduction of true Hox genes as two Hox genes -Hox3 and ftz- lost their homeotic function and obtained new functions in the insect embryo, which is associated with a divergence of the sequence of the gene [2628].

Hox gene duplications have been proposed to be one of the genetic mechanisms behind the diversification of vertebrates [e.g. [29]]. However it remains difficult to draw a direct link between Hox gene duplications and morphological evolution. Recent results from Lynch et al [30] suggest an important role for the action of positive Darwinian selection in the divergence of vertebrate Hox genes after cluster duplications. The locations in the homeodomain of the sites that are under positive selection suggest that they are involved in protein-protein interactions. This suggests that adaptive evolution actively contributed to Hox gene function [30]. Indeed, in the Cs-Ubx-2 homeodomain there are two amino acid exchanges compared to the homeodomain of Cs-Ubx-1 or of Ubx of most other arthropods (Fig 1). Only in the honey-bee and the crustaceans Moina and Artemia there is one amino acid exchange in the homeodomain, in all three cases an A to S exchange at position 37 of the homeodomain (not shown). Also one of the two exchanges in Cs-Ubx-2 is an A to S on position 37. The sequence divergence in the homeodomain of Cs-Ubx-2 thus might be associated with a functional divergence. However the mechanism of the divergence is unknown, leaving open the role of Hox gene duplication in morphological evolution of chelicerates.

Duplicated genes in the spider: a whole genome duplication?

The most important question that comes up now is on the origin of these three duplicated Hox genes in Cupiennius. There are two options. First, they result from a duplication of the complete cluster. This would imply that either additional Hox genes are present as two copies that have not be found so far, or that one copy has been lost for the other Hox genes, as has happened to some of the Hox genes in the duplicated vertebrate clusters. Mammals for instance possess four Hox clusters, but most of the paralogs are not present as four copies as some of them have been lost in some of the clusters [11]. All data for Cupiennius Hox genes were obtained via either PCR approaches or cDNA library screening [15]. As there is no genome project for the spider yet, this means that it is presently unclear whether additional Hox genes are present as duplicated copies in the spider. The second possible explanation for the three duplicated Cupiennius Hox genes could be three independent tandem duplications of the individual genes. Additional analyses are required to identify the genomic organization of the spider Hox genes, and to find out whether these genes are indeed organized in two clusters, or whether the duplicated genes are serial duplications within a single Hox cluster.

However, there is some additional data that point to large-scale duplication of chromosomal fragments or even complete genomes in the spider. So far we also have found in our PCR screens several other genes that are present in two or more copies in the transcriptome of the spider Cupiennius, like extradenticle, homothorax, H15, Wnt5, Wnt7, engrailed, Delta, Suppressor of hairless, Krüppel, runt, pairberry, optomotor blind odd-skipped, apterous, orthodenticle [[19, 3137], our unpublished data]. In contrast, in most other arthropods most of these genes are present as one copy only. The relative high number of duplicated genes may point to a major duplication event in lineage to the spider, which might be caused by a whole genome duplication. A spider genome project would help to verify this.

Two posterior expression boundaries of spider Hox genes

Now data from all ten different arthropod Hox gene classes are known from this spider, another fact becomes obvious, that we already recognized previously based on a smaller data set [38], but which becomes even more prominent by new data on Cs-pb, Cs-Scr, and Cs-ftz [this paper, [19]]. There are two discrete posterior expression boundaries for Hox genes in the Cupiennius (Fig 6). The expression of all anterior Hox genes (lab, pb, Hox3, Dfd, Scr, ftz) ends at the boundary between fourth walking leg (L4) and first opisthosomal segment (O1), which is at the tagma boundary between prosoma and opisthosoma. Also the posterior Hox genes (Antp, Ubx, abdA, AbdB) all have the same posterior expression border: the very posterior end of the embryo. There is only one Hox gene, Cs-Antp, that crosses the tagma boundary (Fig 6). In other arthropods, but also in vertebrates, most of these posterior expression borders are not defined as well as in the spider [12, 39].

The reason for the two discrete posterior expression borders remains unclear and we only can speculate on this. Between L4 and O1 is an important morphological boundary, the one between the two tagmata of the spider: the prosoma and the opisthosoma. The Hox genes might play a role in the specification of this boundary. In contrast, several Hox genes cross tagmata borders in other arthropods [e.g. [12, 23]]. If the Hox genes play a role in tagma border specification, then this must be a peculiarity of the spider.

Another explanation could be that the anterior Hox genes are required for the specification of the different appendages in the spider. All six anterior Hox genes are expressed in distinct patterns within the appendages suggesting a role of them in appendage specification [[15, 17, 19], this paper] (see also Fig 3 and 4). It has been shown that Hox gene expression is associated with morphological diversification of leg segments in insects [40]. Indications for interactions between Hox genes in the spider legs come also from the weaker expression of Cs-Dfd-2 in L3 and L4 that coincides with the stronger expression of Cs-Scr-1, Cs-Scr-2 and Cs-ftz in L3 and L4 (Fig. 3F, Fig. 4C, Fig. 4F and reference [19]. Thus there might be a cross regulation between these Hox genes in the legs. Such a role of these Hox genes in the legs may form the reason for shared posterior expression boundaries (Fig. 6). The border between segments with and without appendages coincides with the tagma boundary between prosoma and opisthosoma. Spiders have true appendages on six segments: the cheliceres, the pedipalps, and four pair of walking legs. The more posterior segments do not have true appendages, however the second to fifth opisthosomal segment develop limb buds that give rise to the respiratory organs and the spinnerets [41].

A third possible explanation could be that the discrete Hox gene expression boundary is a result of the segmentation process that acts more upstream in the regulatory cascade and that lays down the segments. It is known from insects that the segmentation gene cascade indeed also controls the expression of Hox genes [4244]. In insects this is mainly done by orthologs of gap genes. It is not known yet what genes regulate the expression of the spider Hox genes. A number of spider Hox genes obey parasegmental boundaries, as they do in Drosophila [31]. Parasegmental boundaries are important developmental boundaries in the early embryo and are specified by the segmentation gene cascade [45]. Segment-polarity genes like wingless, cubitus interruptus and engrailed maintain the parasegmental boundaries in arthropods. We assume that at least in part the same upstream acting regulatory machinery controls the segment-polarity genes and Hox genes in the spider as their expression boundaries match exactly. The discrete posterior expression border of the spider Hox genes is the result of genes that control them and these therefore may be an output of the upstream segmentation machinery that also control the expression boundaries of the segment-polarity genes. The assumption that the discrete Hox gene expression boundary could be the result of the segmentation process is strengthened by previous work in Cupiennius that suggested that there may be at least partially a difference in the mechanisms that specify the anterior segments and the posterior segments [34]. The posterior segments form sequentially from a posterior growth zone and may be partially regulated in a different way. The discrete boundary of Hox gene expression at the prosoma-opisthosoma boundary therefore could reflect such a difference in the regulation of segmentation between the anterior and the posterior segments.

Methods

Spiders and embryos

Embryos were obtained from our Cupiennius salei Keyserling (Chelicerata, Arachnida, Araneae, Ctenidae) colony in Cologne. Cocoons with embryos were taken from fertilized adult female spiders, RNA was isolated and reverse transcribed into cDNA as described previously [31], embryos were treated and fixed as described previously [17, 31].

Isolation of additional Cupiennius salei Hox genes

Initial PCR fragments of the Cs-pb, Cs-Dfd-2, Cs-Scr-1, and Cs-Scr-2 were obtained using primer combinations directed against sequences in the homeodomain. We used the following primer combinations: for Cs-pb and Cs-Dfd-2 the primers 1521 (5'-GGA TTC TAY CCI TGG ATG-3') and 1520 (5'-CAT ICK ICK RTT YTG RAA CCA-3'), for Cs-Scr the primers 1289 (5'-CCN CAR ATH TAY CCN TGG ATG-3') and 1290 (5'-TT CCA YTT CAT NCG NCK RTT WTG- 3'). Additional sequences have been obtained by subsequent RACE-PCR. Additional sequence of the At-Dfd-1 gene has also been obtained via RACE-PCR, using primers based on a short sequence published by Abzhanov et al [14]. The clones have been sequenced in both directions and the sequences are available under the accession numbers AM419029 to AM419032.

Expression analysis

The expression patterns of the genes have been analyzed by in situ hybridizations using digoxigenin labelled anti-sense RNA probes [17]. We used the following probes: for Cs-Dfd-1, Cs-Ubx-1 and Cs-Ubx-2 we used probes prepared from the cDNA clones isolated from the cDNA library [15] (accession numbers CAA07498, CAA07500, CAA07501), for Cs-pb, Cs-Scr-1 and Cs-Scr-2 we used probes prepared from the all available cDNA sequence (accession numbers AM419029, AM419030, AM419031), for Cs-Dfd-2 we used a probe prepared from a 3'RACE fragment, corresponding to nt 84–1407 of the available cDNA information (accession number AM419032).

Declarations

Acknowledgements

We thank Alistair McGregor for critical reading of the manuscript. This work was supported in part by the DFG via SFB 572 of the University of Cologne and by the European Union via the Marie Curie Research and Training Network ZOONET (MRTN-CT-2004-005624).

Authors’ Affiliations

(1)
Institute for Genetics, University of Cologne
(2)
Department of Developmental Biology, Friedrich-Alexander University Erlangen, Institute for Biology

References

  1. Carroll SB: Homeotic genes and the evolution of arthropods and chordates. Nature. 1995, 376: 479-485. 10.1038/376479a0.View ArticlePubMedGoogle Scholar
  2. McGinnis W, Hart CP, Gehring WJ, Ruddle FH: Molecular cloning and chromosome mapping of a mouse DNA sequence homologous to homeotic genes of Drosophila. Cell. 1984, 38: 675-680. 10.1016/0092-8674(84)90262-9.View ArticlePubMedGoogle Scholar
  3. Lewis EB: A gene complex controlling segmentation in Drosophila. Nature. 1978, 276: 565-570. 10.1038/276565a0.View ArticlePubMedGoogle Scholar
  4. McGinnis B, Krumlauf R: Homeobox genes and axial patterning. Cell. 1992, 68: 283-302. 10.1016/0092-8674(92)90471-N.View ArticlePubMedGoogle Scholar
  5. Carroll SB, Grenier JK, Weatherbee SD: From DNA to diversity, Molecular Genetics and the evolution of animal design. 2005, Malden MA: Blackwell Publishing, 2Google Scholar
  6. Pearson JC, Lemons D, McGinnis W: Modulating Hox gene function during animal body patterning. Nature Reviews Genetics. 2005, 6: 893-904. 10.1038/nrg1689.View ArticlePubMedGoogle Scholar
  7. Chourrout D, Delsuc F, Chourrout P, Edvardsen RB, Rentzsch F, Renfer E, Jensen MF, Zhu B, de Jong P, Steele RE, Technau U: Minimal ProtoHox cluster inferred from bilaterian and cnidarian Hox complements. Nature. 2006, 442: 684-687. 10.1038/nature04863.View ArticlePubMedGoogle Scholar
  8. Cook CE, Jiménez E, Akam M, Saló E: The Hox gene complement of acoel flatworms, a basal bilaterian clade. Evol Dev. 2004, 6: 154-163. 10.1111/j.1525-142X.2004.04020.x.View ArticlePubMedGoogle Scholar
  9. de Rosa R, Grenier JK, Andreeva T, Cook CE, Adoutte A, Akam M, Carroll SB, Balavoine G: Hox genes in brachiopods and priapulids and protostome evolution. Nature. 1999, 399: 772-776. 10.1038/21631.View ArticlePubMedGoogle Scholar
  10. Schubert FR, Nieselt-Struwe K, Gruss P: The Antennapedia-type homeobox genes have evolved from three precursors separated early in metazoan evolution. Proc Natl Acad Sci USA. 1993, 90: 143-147. 10.1073/pnas.90.1.143.PubMed CentralView ArticlePubMedGoogle Scholar
  11. Hoegg S, Meyer A: Hox clusters as models for vertebrate genome evolution. Trends Genet. 2005, 21: 421-424. 10.1016/j.tig.2005.06.004.View ArticlePubMedGoogle Scholar
  12. Hughes CL, Kaufman TC: Hox genes and the evolution of the arthropod body plan. Evol Dev. 2002, 4: 459-499. 10.1046/j.1525-142X.2002.02034.x.View ArticlePubMedGoogle Scholar
  13. Cartwright P, Dick M, Buss LW: HOM/Hox type homeoboxes in the chelicerate Limulus polyphemus. Mol Phyl Evol. 1993, 2: 185-192. 10.1006/mpev.1993.1019.View ArticleGoogle Scholar
  14. Abzhanov A, Popadic A, Kaufman TC: Chelicerate Hox genes and the homology of arthropod segments. Evol Dev. 1999, 1: 77-89. 10.1046/j.1525-142x.1999.99014.x.View ArticlePubMedGoogle Scholar
  15. Damen WGM, Hausdorf M, Seyfarth E-A, Tautz D: A conserved mode of head segmentation in arthropods revealed by the expression pattern of Hox genes in a spider. Proc Natl Acad Sci USA. 1998, 95: 10665-10670. 10.1073/pnas.95.18.10665.PubMed CentralView ArticlePubMedGoogle Scholar
  16. Bastianello A, Ronco M, Burato PA, Minelli A: Hox gene sequences from the geophilomorph centipede Pachymerium ferrugineum (C. L. Koch, 1835) (Chilopoda: Geophilomorpha: Geophilidae): implications for the evolution of the Hox class genes of arthropods. Mol Phylogenet Evol. 2002, 22: 155-161. 10.1006/mpev.2001.1031.View ArticlePubMedGoogle Scholar
  17. Damen WGM, Tautz D: A hox class 3 orthologue from the spider Cupiennius salei is expressed in a Hox-gene-like fashion. Dev Genes Evol. 1998, 208: 586-590. 10.1007/s004270050218.View ArticlePubMedGoogle Scholar
  18. Damen WGM, Tautz D: Abdominal-B expression in a spider suggests a general role for Abdominal-B in specifying the genital structure. J Exp Zool. 1999, 285: 85-91. 10.1002/(SICI)1097-010X(19990415)285:1<85::AID-JEZ10>3.0.CO;2-N.View ArticlePubMedGoogle Scholar
  19. Damen WGM, Janssen R, Prpic N-M: Pair rule gene orthologs in spider segmentation. Evol Dev. 2005, 7: 618-628. 10.1111/j.1525-142X.2005.05065.x.View ArticlePubMedGoogle Scholar
  20. Telford MJ, Thomas RH: Expression of homeobox genes shows chelicerate arthropods retain their deuterocerebral segment. Proc Natl Acad Sci USA. 1998, 95: 10671-10675. 10.1073/pnas.95.18.10671.PubMed CentralView ArticlePubMedGoogle Scholar
  21. Manuel M, Jager M, Murienne J, Clabaut C, Le Guyader H: Hox genes in sea spiders (Pycnogonida) and the homology of arthropod head segments. Genes Evol. 2006, 216: 481-491. 10.1007/s00427-006-0095-2.View ArticleGoogle Scholar
  22. Hughes CL, Kaufman TC: Exploring the myriapod body plan: expression patterns of the ten Hox genes in a centipede. Development. 2002, 129: 1225-1238.PubMedGoogle Scholar
  23. Janssen R, Damen WGM: The ten Hox genes of the millipede Glomeris marginata. Dev Genes Evol. 2006, 216: 451-465. 10.1007/s00427-006-0092-5.View ArticlePubMedGoogle Scholar
  24. Krumlauf R: Hox genes in vertebrate development. Cell. 1994, 78: 191-201. 10.1016/0092-8674(94)90290-9.View ArticlePubMedGoogle Scholar
  25. Lynch M, Conery JS: The evolutionary fate and consequences of duplicate genes. Science. 2000, 290: 1151-1555. 10.1126/science.290.5494.1151.View ArticlePubMedGoogle Scholar
  26. Falciani F, Hausdorf B, Schröder R, Akam M, Tautz D, Denell R, Brown S: Class 3 Hox genes in insects and the origin of zen. Proc Natl Acad Sci USA. 1996, 93: 8479-8484. 10.1073/pnas.93.16.8479.PubMed CentralView ArticlePubMedGoogle Scholar
  27. Telford MJ: Evidence for the derivation of the Drosophila fushi tarazu gene from a Hox gene orthologous to lophotrochozoan Lox5. Curr Biol. 2000, 10: 349-253. 10.1016/S0960-9822(00)00387-0.View ArticlePubMedGoogle Scholar
  28. Damen WGM: fushi tarazu: A Hox gene changes its role. BioEssays. 2002, 24: 992-995. 10.1002/bies.10180.View ArticlePubMedGoogle Scholar
  29. Holland PWH, Garcia-Fernandez J: Hox Genes and Chordate Evolution. Dev Biol. 1996, 173: 382-395. 10.1006/dbio.1996.0034.View ArticlePubMedGoogle Scholar
  30. Lynch VJ, Roth JJ, Wagner GP: Adaptive evolution of Hox-gene homeodomains after cluster duplications. BMC Evol Biol. 2006, 6: 86-10.1186/1471-2148-6-86.PubMed CentralView ArticlePubMedGoogle Scholar
  31. Damen WGM: Parasegmental organization of the spider embryo implies that the parasegment is an evolutionary conserved entity in arthropod embryogenesis. Development. 2002, 129: 1239-1250.PubMedGoogle Scholar
  32. Prpic N-M, Janssen R, Wigand B, Klingler M, Damen WGM: Gene expression in spider appendages reveals reversal of exd/hth spatial specificity, altered leg gap gene dynamics, and suggests divergent distal morphogen signaling. Dev Biol. 2003, 264: 119-140. 10.1016/j.ydbio.2003.08.002.View ArticlePubMedGoogle Scholar
  33. Stollewerk A: Recruitment of cell groups through Delta/Notch signalling during spider neurogenesis. Development. 2002, 129: 5339-5348. 10.1242/dev.00109.View ArticlePubMedGoogle Scholar
  34. Schoppmeier M, Damen WGM: Suppressor of Hairless and Prenesilin phenotypes imply involvement of canonical Notch-signalling in segmentation of the spider Cupiennius salei. Dev Biol. 2005, 280: 211-224. 10.1016/j.ydbio.2005.01.024.View ArticlePubMedGoogle Scholar
  35. Schoppmeier M, Damen WGM: Expression of Pax group III genes suggests a single-segmental periodicity for opisthosomal segment patterning in the spider Cupiennius salei . Evol Dev. 2005, 7: 160-169. 10.1111/j.1525-142X.2005.05018.x.View ArticlePubMedGoogle Scholar
  36. Damen WGM, Saridaki T, Averof M: Diverse adaptations of an ancestral gill: a common evolutionary origin for wings, breathing organs and spinnerets. Curr Biol. 2002, 12: 1711-1716. 10.1016/S0960-9822(02)01126-0.View ArticlePubMedGoogle Scholar
  37. Damen WGM, Weller M, Tautz D: Expression patterns of hairy, even-skipped, and runt in the spider Cupiennius salei imply that these genes were segmentation genes in a basal arthropod. Proc Natl Acad Sci USA. 2000, 97: 4515-4519. 10.1073/pnas.97.9.4515.PubMed CentralView ArticlePubMedGoogle Scholar
  38. Damen WGM, Tautz D: Comparative molecular embryology of arthropods: the expression of Hox genes in the spider Cupiennius salei. Invertebrate Reproduction and Development . 1999, 36: 203-209.View ArticleGoogle Scholar
  39. McGinnis W, Krumlauf R: Homeobox genes and axial patterning. Cell. 1992, 68: 283-302. 10.1016/0092-8674(92)90471-N.View ArticlePubMedGoogle Scholar
  40. Mahfooz NS, Li H, Popadic A: Differential expression patterns of the hox gene are associated with differential growth of insect hind legs. Proc Natl Acad Sci USA. 2004, 101: 4877-4882. 10.1073/pnas.0401216101.PubMed CentralView ArticlePubMedGoogle Scholar
  41. Foelix RF: Biology of spiders. 1996, New York: Oxford University Press, 2Google Scholar
  42. Wu X, Vasisht V, Kosman D, Reinitz J, Small S: Thoracic patterning by the Drosophila gap gene hunchback. Dev Biol. 2001, 237: 79-92. 10.1006/dbio.2001.0355.View ArticlePubMedGoogle Scholar
  43. Bucher G, Klingler M: Divergent segmentation mechanism in the short germ insect Tribolium revealed by giant expression and function. Development. 2004, 131: 1729-1740. 10.1242/dev.01073.View ArticlePubMedGoogle Scholar
  44. Cerny AC, Bucher G, Schröder R, Klingler M: Breakdown of abdominal patterning in the Tribolium Krüppel mutant jaws. Development. 2005, 132: 5353-5363. 10.1242/dev.02154.View ArticlePubMedGoogle Scholar
  45. Martinez-Arias A, Lawrence PA: Parasegments and compartments in the Drosophila embryo. Nature. 1985, 313: 639-642. 10.1038/313639a0.View ArticlePubMedGoogle Scholar

Copyright

© Schwager et al; licensee BioMed Central Ltd. 2007

This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Advertisement