Mannaert et al. BMC Genomics 2010, 11:658 http://www.biomedcentral.com/1471-2164/11/658 R E S E A R C H A R T I C L E Open Access Comparative analyses of vertebrate posterior HoxD clusters reveal atypical cluster architecture in the caecilian Typhlonectes natans An Mannaert1, Chris T Amemiya2, Franky Bossuyt1* Abstract Background: The posterior genes of the HoxD cluster play a crucial role in the patterning of the tetrapod limb. This region is under the control of a global, long-range enhancer that is present in all vertebrates. Variation in limb types, as is the case in amphibians, can probably not only be attributed to variation in Hox genes, but is likely to be the product of differences in gene regulation. With a collection of vertebrate genome sequences available today, we used a comparative genomics approach to study the posterior HoxD cluster of amphibians. A frog and a caecilian were included in the study to compare coding sequences as well as to determine the gain and loss of putative regulatory sequences. Results: We sequenced the posterior end of the HoxD cluster of a caecilian and performed comparative analyses of this region using HoxD clusters of other vertebrates. We determined the presence of conserved non-coding sequences and traced gains and losses of these footprints during vertebrate evolution, with particular focus on amphibians. We found that the caecilian HoxD cluster is almost three times larger than its mammalian counterpart. This enlargement is accompanied with the loss of one gene and the accumulation of repeats in that area. A similar phenomenon was observed in the coelacanth, where a different gene was lost and expansion of the area where the gene was lost has occurred. At least one phylogenetic footprint present in all vertebrates was lost in amphibians. This conserved region is a known regulatory element and functions as a boundary element in neural tissue to prevent expression of Hoxd genes. Conclusion: The posterior part of the HoxD cluster of Typhlonectes natans is among the largest known today. The loss of Hoxd-12 and the expansion of the intergenic region may exert an influence on the limb enhancer, by having to bypass a distance seven times that of regular HoxD clusters. Whether or not there is a correlation with the loss of limbs remains to be investigated. These results, together with data on other vertebrates show that the tetrapod Hox clusters are more variable than previously thought. Background Perhaps the best studied gene clusters in animals are the Hox clusters, not only for their importance in the estab- lishment of the metazoan body plan, but also for their tight genomic organization. Hox genes encode transcrip- tion factors that belong to the family of homeodomain proteins and play an essential role in the establishment of the anterior-posterior body axis during embryonic development. In addition, they are also involved in * Correspondence: fbossuyt@vub.ac.be 1Biology Department, ECOL, Amphibian Evolution Lab, Vrije Universiteit Brussel, Brussels, Belgium Full list of author information is available at the end of the article patterning of limbs and in organogenesis [1-4]. In vertebrates, the expression domains of Hox genes are collinear in space and time, and reflect their chromoso- mal arrangement [5]. Invertebrates possess one - often interrupted or disin- tegrated - Hox cluster, while all vertebrates have multi- ple clusters [6-8]. Gnathostomes typically have four Hox clusters that arose by subsequent duplications in the stem lineage of vertebrates, while most ray-finned fishes contain seven (e.g. zebrafish) to thirteen (e.g. salmon) clusters as a result of additional, teleost-specific genome duplications and subsequent cluster losses [9-11]. Due to these additional duplications, the gene content of the © 2010 Mannaert et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.