Mannaert et al. BMC Genomics 2010, 11:658 http://www.biomedcentral.com/1471-2164/11/658 Page 6 of 9 horn shark zebrafish coelacanth caecilian frog anole lizard chicken opossum human 2 1 3 20 4 26 5 31 7 8 9 1170 18 22 Hoxd-14 2332 loss Hoxd-13 loss Hoxd-12 loss 11 12 15 13 27 33 29 30 7 31 7 18 91518 6 28 14 16 19 21 24 25 7 11 17 29 13 11 11 6 14 1619 21 14 500 400 300 200 100 0 mya Figure 3 Reconstruction of vertebrate posterior HoxD cluster evolution. A Dollo parsimony reconstruction of the evolution of HoxD cluster elements shows the ancestral presence of 16 conserved elements in jawed vertebrates. Divergence times of the species are taken from Alfaro et al. [73]. A schematic representation of the posterior HoxD cluster with correct relative distances is shown on the right. The human cluster is representative for placental mammals, except for the loss of footprint 14, which did not occur in other mammals. Potential ncRNAs are symbolized by orange ovals, putative cis-regulatory elements by blue ovals. Gain of elements is shown as full ovals, with the respective footprint number underneath, open ovals and bars represent the loss of a footprint and Hoxd gene, respectively. Ovals with dotted lines denote equivocal reconstruction of footprint 11, which is due to missing data in the chicken sequence. Gains and losses of footprints were not dated. the amphibian ancestor: fp9, which is one of two motifs of RXII, and fp15, a small footprint in the 3’ UTR of Hoxd-13. The conserved region RXII is a boundary ele- ment located in the intergenic region between Evx-2 and Hoxd-13, whose promoters are in each other’s vici- nity. Unlike Hoxd genes, Evx-2 is expressed in the cen- tral nervous system, regulated by the neural enhancer of the GCR. RXII is considered to function as an insulator to prevent the ectopic expression of Hoxd genes in the nervous system, since GCR regulation is not promoter- specific [23]. Although tracker failed to recover the entire RXII element (fp9 and fp10) in the zebrafish, short, apparently homologous sequences were found in the zebrafish cluster in the same area as in other verte- brates [23]. Moreover, both footprints were found in the horn shark, which points to their ancestral presence in vertebrates, and therefore we conclude that fp9 was lost in amphibians. Footprint 15 is a short DNA stretch of ~22 bp with a highly conserved motif of 7 bp located in the 3’ UTR of Hoxd-13. This motif is somewhat degen- erated in the coelacanth, which does not have a func- tional Hoxd-13 gene, and it is located around 14 kb 5’ to ψHoxd-13, which may indicate that this sequence is not homologous to the other vertebrate fp15 sequences. On the other hand, pseudogenization of Hoxd-13 and insertion of repetitive sequences may have promoted the relative relocation of this element. For our reconstruction, we considered all footprints as identified by the tracker software to be truly conserved sequences. Therefore, we conclude that this element originated in Sarcopterygii, and was lost in amphibians. In theory, this footprint can serve as a target for the microRNA miR- 26, which is expressed in neurons and astrocytes of the developing mouse brain [65]. The absence of both elements may indicate that restriction of Hoxd-13 expression in neural tissue is regulated differently in amphibians. Conclusion The posterior HoxD cluster of the caecilian is much larger than that of most known vertebrates due to the accumulation of interspersed and inverted repeats accompanied with Hoxd-12 gene loss. A similar event occurred in the coelacanth and in the corn snake HoxD cluster. Whether these occurrences are reflected in the morphology of these species is not clear, but it is