Runt Gene Evolution Figure 2. Overview of the Runt gene evolution in chordates. The stepwise evolution of cartilage and bone and the most likely time intervals of Runt gene duplications (Dup) are indicated. The position of tunicates is contentious [31] which is indicated by a dashed line. In this context it is of interest that pre-neural crest cells have been observed in tunicates [60]. doi:10.1371/journal.pgen.1000025.g002 Runt gene expression in skeletal elements of hagfish and dogfish To determine a possible role for Runt genes in the skeleton we asked the question if Runt genes are expressed in skeletal elements of hagfish. Using quantitative Reverse Transcriptase PCR (qRT- PCR) from dissected tissues we found that the MgRunxA gene had its highest expression in hard cartilage, followed by the gill region and soft cartilage (Figure 3). Compared to the MgRunxA gene the MgRunxB gene was only weakly expressed with the strongest expression in the gill region. In situ hybridizations confirmed the high expression of MgRunxA in hard cartilage (Figure 3B and 3C). In adult dogfish the Runt genes show ubiquitous expression but it is noteworthy that all Runt genes had their third highest expression in the gill gut cartilage. For all three dogfish Runt genes the highest expression was found in the skin (Figure 4). We performed in situ hybridization to characterize the distribution of Runt expression in the skin (Figure 5A–5C). All three Runt genes were expressed in the placoid scales in the skin of dogfish embryos. ScRunx1 and ScRunx3 were expressed in the basal epidermis cells of the stratum germinativum, whereas ScRunx2 was found at the site where later the basal plate will develop. Based on the similarities between scales and teeth we performed expression analysis of Runt genes in the developing teeth of dogfish embryos. In the developing teeth Figure 3. Analysis of hagfish MgRunxA and –B expression in different tissues of adult animals. Quantification of MgRunxA and –B expression by qRT-PCR (A). Whereas MgRunxB was only weakly expressed in all tissues analyzed, MgRunxA showed a strong expression in calcified cartilage gills and soft cartilage. Expression of MgRunxA was also detected by radioactive in situ hybridisation in hard cartilage tissue (B, C). Insert of (B) is shown at higher magnification in (C) displaying the silver grains of the autoradiography emulsion indicating MgRunxA expression. B: Brain, C-h: Hard cartilage, C-s: Soft cartilage, Cho: Chorda, G: Gills, Gb: Gall bladder, G-a: Anterior gut, G-m: Midgut, G-h: Hindgut, H: Heart, L: Liver, Mu: Muscle, Sk: Skin. doi:10.1371/journal.pgen.1000025.g003 PLoS Genetics | www.plosgenetics.org 4 March 2008 | Volume 4 | Issue 3 | e1000025