Figure 9. Runt dependent regulation of the B. floridae Hh promoter. (A) Scheme of the BfHh promoter with putative Runt binding sites. Number and position relative to the transcription start site is given. (B) Electrophoretic mobility shift assays using oligos containing R1–R6 Runt binding sites. BlRunt can bind to each of the putative binding sites. Strongest binding is observed for the oligo with the closely adjacent binding sites R5 and R6 and for the R1 oligo. Nuclear extracts without BlRunt do not show a mobility shift of the oligos (data not shown). (C) Runt dependent activation of the BfHh promoter in NIH3T3 cells. Overexpression of either BlRunt or MmRunx2 leads to activation of the indicated promoter constructs compared to constructs co-transfected with an empty expression vector. doi:10.1371/journal.pgen.1000025.g009 evolving Runx2 gene suggests that during chordate evolution the entire Runt locus was triplicated. Runt genes and the evolution of cartilage and bone in vertebrates Cartilage has evolved multiple times in metazoa [1]. Here we focus on the vertebrate cellular cartilage expressing Col2a1 as the predominant matrix protein. Differentiation of this cartilage is regulated by a molecular network including Sox9, a transcription factor that directly regulates Col2a1 expression [36]. Furthermore Sox9 is a target of PTH related protein (PTHrP) that controls chondrocyte differentiation through a negative feedback loop with Indian hedgehog (Ihh). Runx2 in turn directly regulates Ihh [10]. Besides Runx2 also Runx1 and Runx3 genes are expressed during murine and zebrafish cartilage formation. However Runx2 and Runx3 appear to be the most important Runt genes for skeletogenesis [6,23]. In hagfish soft and hard cartilage can be distinguished [2] and a Col2a1-homologous protein is expressed only in soft cartilage [4]. It is unknown if a protein homologous to Col1a1 is expressed in hard cartilage as it is the case in mammalian bone. As shown in Runt Gene Evolution Figure 3 the hagfish MgRunxB gene is only weakly expressed in both types of cartilage. However, the MgRunxA gene had its strongest expression in hard cartilage and its third highest expression in soft cartilage (Figure 3A). We only analyzed tissues from adult hagfish of medium size (30–40 cm). The fact that hagfish grow up to a length of 80 cm suggests that the Runt gene expression in hagfish cartilage is also of importance for the growth of the skeleton. The view that Runt genes have a conserved functional role in skeletogenesis is also supported by our finding of Runt gene expression in the developing cartilage of dogfish. We detected a strong expression in visceral cartilage for all three dogfish Runt genes by qRT-PCR (Figure 4). Furthermore we performed in situ hybridizations on dogfish embryos and found ScRunx2 to be expressed in the cartilage of the fin and together with ScRunx3 in the gill gut cartilage (Figure 6). In lamprey (another representative of jawless vertebrates) the Col2a1 gene is expressed in cartilage along with Sox9 and PTHrP, indicating that they were already a part of the chondrogenic gene repertoire in early vertebrate evolution [3]. Our finding of dogfish and hagfish Runt expression in cartilage together with the well- known role of Runt genes in skeletogenesis, suggests that Runt genes can now be considered to be a part of the ancient molecular machinery for cartilage formation in the stem species of vertebrates. ScRunx1–3 gene expression in teeth and placoid scales Placoid scales are small conical structures in the skin of cartilaginous fish. We found that all three dogfish Runt genes are expressed in the developing placoid scales (Figure 5A–5C). Interestingly, the basal plate of scales and teeth is initiated by osteoblasts which continue to secrete bone matrix in a basal direction, while slightly later, the odontoblasts secrete dentine on the pulpar side on the basal plate [37]. Since ScRunx2 is expressed in the developing basal plate it is an interesting speculation that the expression of Runx2 at this site might reflect the origin of bone as a dermal tissue in early vertebrate evolution. The dermoskeleton is the first to show mineralization in vertebrate phylogeny [38]. This mineralized dermoskeleton was composed of odontodes (dermal ‘‘teeth’’) supported by extensively developed bone, imposing mineralization upon the collagenous layer of the dermis [38]. In placoid scales as well as in the developing teeth ScRunx1 and ScRunx3 were expressed in the stratum germinativum, whereas ScRunx2 was found at the site where later the basal plate will develop (Figure 5A–5F). In mammals teeth develop as epithelial appendages in which sequential and reciprocal interaction between the ectoderm and underlying neural crest derived mesenchyme constitute a central developmental mechanism [1,14]. The dental epithelial cells differentiate into ameloblasts and mesenchymal cells into odontoblasts, secreting the matrices enamel, and dentin respectively [1]. Runx2 and Runx3 expression is confined to mesenchymal tissues, whereas Runx1 was found to be restricted to epithelia [14]. According to a classical view teeth evolved secondarily from skin denticles moving into the mouth (reviewed in [39]). However, this model was recently challenged by the proposal that sets of denticles on the pharyngeal (gill) arches and not external denticles were the precursors of the organized tooth families [39]. This alternative theory was based on the observations of homologous arrays of denticle whorls occurring within the pharyngeal region of jawless fish such as the thelodont Loganellia [40]. In this model the endoderm played an important role in the patterning process involved in the production of denticles on the postbranchial lamina [39]. It was assumed that the denticles on the postbranchial PLoS Genetics | www.plosgenetics.org 9 March 2008 | Volume 4 | Issue 3 | e1000025