Runt Gene Evolution Figure 5. ScRunx1–3 expression analysis by in situ hybridization in placoid scale (A–C) and tooth development (D–F). Bright field is given on top, dark field below. ScRunx1 (A, D) and –3 (C, F) are expressed in the basal epidermis cells of the stratum germinativum, which forms the enamel organ, whereas ScRunx2 (B, E) is found at the site of the developing basal plate. These expression patterns were identical in teeth and placoid scales. (G) Scheme of Runt expression in placoid scales and teeth with overlapping expression of ScRunx1 and –3 in the stratum germinativum (light grey) and ScRunx2 in the developing basal plate (dark grey). Dotted lines represent section planes of transverse sections in (A,C–F). Section in (B) is a longitudinal section. doi:10.1371/journal.pgen.1000025.g005 A chordate stem species with only a single Runt gene is the most parsimonious assumption since the genomes of cnidarians, nematodes, cephalochordates and tunicates harbour also only a single Runt gene. The presence of two Runt genes in sea urchin is most likely a result of a tandem duplication, as we found both genes on a single genomic contig and they cluster together in our phylogenetic analysis (Figure 1). It was recently postulated that tunicates and not cephalochordates are the sistergroup of vertebrates [31,32]. Focusing on our aim to reconstruct the framework for Runt gene evolution, both alternative taxonomic PLoS Genetics | www.plosgenetics.org 6 March 2008 | Volume 4 | Issue 3 | e1000025