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).
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
EES and MS carried out the cloning of the Cs genes and analysis of the expression patterns. MP recovered the complete At-Dfd-1 sequence and helped with the sequence analysis. WGMD conceived of the study, participated in its design and coordination, and wrote the manuscript. All authors read and approved the final manuscript.
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).
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