Results
- Duplicated Hox genes in the spider Cupiennius salei

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.

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.

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).

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).

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).