Mannaert et al. BMC Genomics 2010, 11:658 http://www.biomedcentral.com/1471-2164/11/658 mouse and human. In the caecilian, the 5’ end of the HoxD cluster, starting from the stop codon of Evx-2, situated on the reverse strand, until the stop codon of Hoxd-10 is 107 kilo base pairs (kb), which is over 2.5 times larger than the orthologous region in mammals (approximately 40 kb) (Figure 1). Apart from the caeci- lian, the coelacanth and the lizard sequences are also larger than average, with lengths of 82 kb and 95 kb, respectively. The expansion of the caecilian HoxD clus- ter is mainly due to the lengthening of the intergenic region between Hoxd-13 and Hoxd-11, which is over six times larger than in the human. In the coelacanth, the intergenic region between Evx-2 and Hoxd-12, where the Hoxd-13 gene was lost, is almost four times longer than the corresponding region in mammals. In the anole lizard, not only the HoxD cluster, but also the other three Hox clusters are significantly longer [15]. Global alignment with the other vertebrates uncovered the remains of Hoxd-12 in the caecilian (Figure 2). The presence of multiple frameshift-producing indels and stop codons implies that it is no longer protein coding and has become a pseudogene (ψHox-d12). As frogs and caecilians comprise the basal split within amphibians [32], the most parsimonious explanation for the absence of Hoxd-12 from the Silurana tropicalis genome and its pseudogenization in the caecilian is an early loss in amphibian evolution with unequal rates of evolution in T. natans and S. tropicalis, although two independent loss events cannot be excluded. We also discovered a pseudogene (ψHoxd-13) in the coelacanth posterior HoxD sequence. A blastx analysis revealed fragments of both exons that could still be aligned with other verte- brate Hoxd-13 genes. The length of the coding sequences of Hox genes is similar in both amphibian species, except for Hoxd-13, which is over 100 bp shorter in Typhlonectes. In general, most posterior HoxD coding sequences have comparable caecilian human Page 3 of 9 Evx-2 d13 d12 d11 d10 shark zebrafish coelacanth caecilian frog lizard chicken opossum dog human 10 kb 20 kb 30 kb 40 kb 50 kb Figure 2 Global alignment of the vertebrate posterior HoxD clusters. The graphs represent the percentage of nucleotide sequence conservation of each vertebrate HoxD cluster compared to the orthologous region in the mouse HoxD cluster. Blue peaks represent coding regions, corresponding to the Hox exons on top of the figure; pink peaks represent highly conserved non-coding sequences. The shaded boxes indicate a Hoxd-13 pseudogene in the coelacanth and a Hoxd-12 pseudogene in the caecilian. lengths in all species used in this study, except for Hoxd-13 and Hoxd-11, which are about 25% longer in amniotes and placental mammals, respectively. The amniote or mammalian expansion of the HOXD13 and HOXD11 proteins can be attributed to sequences rich in alanine, glycine, serine and proline. In HOXD13, a homopolymeric repeat consisting of nine alanines in the chicken and 15 alanines in mammals [28] is not present Evx-2 d13 ψd12 d11 d10 Evx-2 d13 d12 d11 d10 0 10 20 30 40 50 60 70 80 90 100 110 120 kb Figure 1 Posterior HoxD cluster architecture of the caecilian. The homologous region of the human is drawn for comparison. The exons of the genes are characterized by black boxes above the cluster (pseudogene in grey); arrows indicate the direction of transcription. The triangles below the cluster represent conserved non-coding regions (putative cis-regulatory elements in blue, potential ncRNA genes in orange) identified by the tracker program, bars and rectangles below the clusters denote repeats. Interspersed repeats identified by Censor are colored dark grey, other repeats, i.e. inverted and direct repeats that are not a part of known transposable elements, are light grey. All distances are drawn to scale.