Mannaert et al. BMC Genomics 2010, 11:658 http://www.biomedcentral.com/1471-2164/11/658 fish Hox clusters is variable, with different gene losses in different species examined. In contrast, the Hox comple- ment of tetrapods is rather conserved, with the same genes present in mammalian and bird genomes [7]. In the genome of the frog Silurana tropicalis, at least one and possibly two genes have been lost [12,13]. Hox gene clusters in vertebrates are compact (around 100 kb in mammals and even shorter in teleost fishes), with highly conserved distances between paralogous genes, and with little or no interspersed repetitive DNA elements [14]. The only exceptions known so far are squamate reptiles, with the lizard Anolis carolinen- sis as a striking example of having accumulated a sub- stantial number of retrotransposons in its Hox clusters, resulting in considerably larger cluster sizes [15,16]. In general, the tight clustering of the Hox genes in verte- brates may be the result of an evolutionary constraint to keep the genes in close proximity, thus maintaining the intergenic distances and prohibiting insertion of interspersed repeats [17]. This constraint may be facili- tated by the presence of cis-regulatory elements within the clusters that are shared by neighboring genes, as well as by remote enhancers producing regulatory landscapes that would be broken when the clusters split [18]. In this context, a suite of global long-range enhancers that control the expression of six genes located 5’ of the HoxD cluster of fishes and mammals has been discovered [19]. This Global Control Region (GCR) reinforces the effect of another enhancer, Prox, that drives the expression of the genes Lnp and Evx-2 - both adjacent to the 5’ end of the HoxD cluster - and the posterior Hoxd genes (Hoxd-13 to Hoxd-10) in the distal limb and genital buds (digit enhancer); it also regulates the expression of Lnp and Evx-2 in the cen- tral nervous system (neural enhancer) [19-21]. The action of the GCR in the nervous system is somehow restricted by boundary elements between Evx-2 and Hoxd-13, while in the limb bud its effect decreases progressively with distance from the 5’ end of the HoxD cluster [22-24]. Defects in Hoxd genes or gene regulation often have an effect on limb development. For example, the mouse Ulnaless mutation causes reduction of the zeugopod, which is the result of the alteration of Hoxd gene expression due to an inversion of the HoxD cluster and subsequent change of cis-regulatory control [19,25,26]. Similar phenotypes can be observed in human meso- melic dysplasia patients, which results from micro- duplications in the HoxD cluster [27]. Normal limb development can also be affected by mutations within Hoxd genes. For example, the expansion of a poly-ala- nine tract in HOXD13 results in the synpolydactyly syn- drome, with abnormal reductions, duplications and fusions of digits [28]. Page 2 of 9 Despite their differences in limb types, frogs and salamanders are characterized by the presence of only four fingers. Of all amphibians, caecilians (Gymno- phiona) probably form the most enigmatic order, as most of them spend their life hidden under the ground. Similar to snakes, they have an elongated trunk and have undergone secondary loss of limbs. The diversity in body plan and limbs among the three amphibian orders may have been affected by changes in Hox gene sequence, or, more likely, regulation (e.g. [29]). At present, the only amphibian genome sequence pub- licly available is from the frog Silurana tropicalis. We constructed a BAC library of the aquatic caecilian Typh- lonectes natans to obtain the sequence of the posterior end of the HoxD cluster, including Evx-2, i.e. the part of the cluster that is important in limb development and which has been shown to be under control of the Global Control Region. Comparative analyses of this region with the orthologous region of other vertebrates and the subsequent identification of conserved, non-coding, putative regulatory elements may shed light on the evolution of the caecilian body plan. Results and Discussion BAC sequencing and annotation The haploid genome size of Typhlonectes natans was estimated at 13.37 pg by flow cytometry with chicken erythrocyte nuclei as an internal standard (data not shown). A pooled BAC library of about 460,000 clones with an average insert size of 107 kb from Typhlonectes natans was constructed, comprising a theoretical 3.8 × coverage of the genome, and screened by PCR to isolate a clone that contained the posterior HoxD cluster. A single clone with an estimated size of approximately 115 kb containing Hoxd-13 was sequenced using 454 sequencing technology [30] and over 15,000 reads were assembled into two supercontigs. The orientation of the two supercontigs was determined by sequencing of both BAC ends, and they were assembled into one final con- tig with a small gap arbitrarily set at 100 base pairs (bp) because the total sequence length is consistent with the estimated insert size. The total caecilian sequence com- prises 116,633 bp, including a 100 bp gap (GenBank HQ398255). An initial blastx analysis and the software GenomeScan [31] identified the genes Evx-2, Hoxd-13, Hoxd-11 and a large part of Hoxd-10. The exon-intron boundaries were refined manually by alignment with Hox sequences of other vertebrates. Hoxd-12 was not found in these analyses. Vertebrate posterior HoxD cluster comparison We compared the posterior HoxD cluster of the caecilian with the clusters of the horn shark, zebrafish, coelacanth, frog, chicken, anole lizard, opossum, dog,