Mannaert et al. BMC Genomics 2010, 11:658 http://www.biomedcentral.com/1471-2164/11/658 in any other species. HOXD13 proteins are overall rich in serine and alanine in mammals, but not in non- amniotes. In addition to the conservation of both exons of each Hox gene, evolutionarily conserved regions were also detected in non-coding intergenic and intronic sequences. These conserved, non-coding sequences or phylogenetic footprints may represent regulatory ele- ments and non-coding RNA genes and were further investigated. Distribution of repeats To investigate whether the caecilian HoxD cluster expansion was accompanied by an accumulation of repeats, we compared it to the Repbase database [33] of known vertebrate repetitive elements using RepeatMas- ker [34] and Censor [35]. RepeatMasker identified 4.74% of the sequence as part of transposable elements, while Censor reports 7.45% interspersed repeats, mostly retro- transposons like LINES and SINES. It must be noted that the hits are not always very strong because of short alignment length. However, blastn searches of all the repeats resulted in a significant match with known repeats (e-value ≤ 1e-05) for 11 out of 54 repeats. More- over, blastx searches of the intergenic regions recovered one additional SINE and one LINE. The majority of the repeats is located in the intergenic region between Hoxd-13 and ψHoxd-12 (Figure 1). The same analyses were performed for the other vertebrate sequences by comparison with the repeat databases for the respective species, if available. In the caecilian and the coelacanth, the repeats are almost exclusively found in the large intergenic region where the pseudogene is situated, i.e. between Hoxd-13 and ψHoxd-12 in the caecilian and between Evx-2 and ψHoxd-13 in the coelacanth. In the lizard and zebrafish, transposable elements are dispersed over the cluster, with a concentration of repeats between Evx-2 and Hoxd-13 in the latter. Additionally, a self-self blastn analysis was performed to identify direct and inverted repeats that are not part of known transposons. This revealed the presence of 30 inverted repeats (stem- loop), 15 palindromes (stem) and 12 direct repeats (minimum identity of 70%, e-value ≤1e-5) in the caeci- lian HoxD cluster. Again, almost all repeats are located in the region between Hoxd-13 and the ψHoxd-12. Some of these repeats are a part of transposable ele- ments, but the majority is unknown. A similar result was obtained for the coelacanth sequence. Here, no direct repeats, 21 inverted repeats and 11 palindromes were found, all located in the region between Evx-2 and Hoxd-12, with several large stretches in the former intron of ψHoxd-13. The anole lizard sequence also con- tains a large amount of inverted and direct repetitive sequences, but spread across the cluster, which is in concordance with the distribution of transposable Page 4 of 9 elements. Few or no additional repeats were found in the other vertebrate HoxD sequences. The presence of interspersed, repetitive DNA may undermine genomic stability [36]. In vertebrate genomes, regions containing developmental genes, such as Hox clusters, are usually devoid of transposable elements [37], suggesting the presence of a constraint against the invasion of foreign elements into a region essential for development. The caecilian, coelacanth, anole lizard and zebrafish have a higher than average amount of repetitive elements in the posterior HoxD cluster, and the caecilian and coela- canth also experienced the loss of a functional Hox gene. Whether the presence of repeats attributed to the pseudogenization of Hoxd-12 in the caecilian (and of Hoxd-13 in the coelacanth), or whether the loss of Hoxd-12 weakened the constraint and allowed repeats to accumulate, remains unknown. In the human gen- ome, pseudogenes are frequently found in the vicinity of long inverted repeats [38]. Despite being essential for proper embryonic develop- ment, the loss of one Hox gene does not necessarily have a negative impact on an organism’s phenotype, as Hox genes can be functionally equivalent [39]. However, expansion of the cluster may have an effect on gene reg- ulation. Since all the genes in this region of the HoxD cluster are under the control of the long-range enhancer GCR, the loss of Hoxd-12 accompanied with the enlar- gement of the distance between Hoxd-13 and Hoxd-11 and the accumulation of repetitive elements in the cae- cilian may have an influence on the effect of the GCR on the expression of Hoxd-11 and Hoxd-10. Whether this effect, if any, is reflected in morphology, is unknown, but it is worth mentioning that a similar clus- ter architecture was found in the corn snake [16]. Some transposable elements have been exapted to modulate gene regulatory networks (reviewed by [40]). As changes in - especially developmental - gene regulation may lead to morphological changes [41], the adoption of a highly derived body plan, such as in caecilians, may have been facilitated by transposable elements. It is possible that caecilians and snakes have employed a similar mechan- ism of limb loss, though this is probably not the case in other limbless squamates, since Hoxd-12 is present in the slowworm Anguis fragilis [16]. Identification of phylogenetic footprints More and more regulatory sequences and non-coding RNA (ncRNA) genes are being discovered in the portion of the genome that does not code for proteins [42]. Although it has been shown that not every regulatory sequence is evolutionarily conserved and that an apparent function cannot always easily be allocated to a conserved sequence [43,44], screening genomes for evo- lutionarily conserved non-coding sequences is a widely