Theria-Specific Homeodomain and cis-Regulatory Element Evolution of the Dlx3–4 Bigene Cluster in 12 Different Mammalian Species†

The genetic basis for the acquisition and retention of innovative morphological structures is of wide interest to evolutionary biologists. Mammals possess specific anatomical features that are not found in other vertebrates, such as external ears, placenta, and hair. It has been inferred that genetic changes in developmental regulatory pathways may contribute, in part, to the evolution of such innovations. In particular, duplicated developmental genes (paralogs) play an important role in generating variations in evolutionarily important morphological structures via deployment of novel cis-regulatory elements. The use of comparative genomics together with mouse transgenic methods provides an effective strategy for interrogating the regulatory landscape of duplicated loci of major developmental genes, as reported here for one of the Dlx gene clusters. The Distal-less (Dlx) genes comprise a homeobox-containing multigene family. The mammalian Dlx gene family consists of six members that are orthologous to the Drosophila Distal-less gene and which are organized into three convergently transcribed (tail-to-tail) bigene clusters; it is presumed that the ancestral bigene cluster was generated initially by tandem gene duplication followed by whole genome duplications (Stock et al., ‘96). Bigene cluster pairs are Dlx1 and Dlx2, Dlx6 and Dlx5, and Dlx41 and Dlx3 (Simeone et al., ‘94; McGuinness et al., ‘96; Nakamura et al., ‘96; Ellies et al., ‘97b; Zerucha and Ekker, 2000), each of which is originally linked to a HOX cluster (HOXD, HOXA, and HOXB, respectively). Dlx expression patterns and functional studies by targeted Dlx deletions provide strong evidence that these transcription factors play major roles in patterning of multiple developmental structures (Merlo et al., 2000; Quint et al., 2000; Zerucha and Ekker, 2000; Zerucha et al., 2000; Depew et al., 2002; Panganiban and Rubenstein, 2002). Though vertebrate Distal-less genes were originally described as being primarily “head” homeobox genes (Duboule, ‘94), they have been shown also to be involved in development of many other structures, that is, limbs, peripheral neurons, whiskers/hair follicles, otic vesicles, teeth, median fins in zebrafish, cement glands in Xenopus, keratinocytes in Xenopus and mice, placenta, genital tubercle and hematopoietic cells in humans (Dirksen et al., ‘93; Akimenko et al., ‘94; Morasso et al., ‘94, ‘99; Porteus et al., ‘94; Shimamoto et al., ‘97; Zerucha et al., ‘97; Park and Morasso, ‘99; Hwang et al., 2008). cis-Regulatory elements confer cell and tissue specificity with regard to the transcriptional machinery and have been implicated as being important for morphological evolution, particularly in the context of embryonic transcription factor genes (Shashikant et al., ‘98; Carroll, 2008; Cretekos et al., 2008; Levine, 2010). Since Dlx genes have been co-opted recurrently in vertebrate evolution by acquisition of novel expression sites, their cis-regulatory element functionality and evolutionary history are of great interest in terms of bauplan divergences in mammals. Delineation of the genomic organization and overlapping expression patterns of the Dlx genes provides a window into understanding the genomic control of development of the many structures described above. Expression patterns of linked Dlx genes have suggested that cis-regulatory elements controlling spatio-temporal expression exist within their respective intergenic regions (Ellies et al., ‘97a). Functional analyses of the intergenic region between paired Dlx genes showed that this region indeed harbors important cis-regulatory elements (Zerucha et al., 2000; Sumiyama et al., 2002; Ghanem et al., 2003). In the Dlx3–4 cluster there are several evolutionarily conserved elements within about 20 kb of the intergenic non-coding region of human and mouse. Moreover, one of these was shown to be a vital enhancer element for branchial arch expression in a Dlx3 specific manner (Sumiyama and Ruddle, 2003). Global alignments using genomic sequences from multiple species is an effective means to identify potentially functional elements in the non-coding regions. The ENCODE project report has shown that a total of 5% of the bases in the genome can be confidently identified as being under evolutionary constraint in mammals, and that approximately 60% of these bases are biologically functional on the basis of experimental assays (ENCODE, 2007). The purpose of the current study was to investigate the rate of change of molecular evolution of cis-elements possibly related to mammalian specific characters of the Dlx3–4 bigene cluster using an extended dataset of 12 diverse mammalian species, including a marsupial and a monotreme. Large-scale sequence comparisons and subsequent constrained motif extraction were conducted with the aim of identifying molecular evolutionary trends in the respective elements. The analyses show that conserved nucleotide elements in intergenic region evolve at markedly different rates and highlight varying patterns of evolutionary divergence among the species. In transgenic mice experiments, we demonstrated that I37-2 cis-regulatory element for branchial arches showed theria-specific conservation and enhancer activity, whereas that of the platypus showed no enhancer activity in branchial arches. The findings serve as guideposts for our ultimate goal to examine the nature in which the cis-regulatory modules in the Dlx bigene clusters act to bring about developmental and gross morphological changes in vertebrate evolution.