Runt Gene Evolution Figure 6. Expression of ScRunx2 and –3 in developing dogfish cartilage. Expression of ScRunx2 was detected in developing cranial and gill bar cartilage (A) and in the proximal cartilage elements of the pectoral fin (B). Expression of ScRunx3 was detected in developing visceral cartilage (C). Cc: cranial cartilage, gb: gill gut cartilage, fc: fin cartilage. doi:10.1371/journal.pgen.1000025.g006 positions of tunicates and lancelets would be consistent with our hypothesis that the stem species of chordates harboured only a single Runt gene. In accordance with the evidence for at least one genome wide duplication, 350 to 650 million years ago [33,34] we detected in dogfish (as a representative of the jawed cartilaginous fish) three Runt genes, orthologous to Amniota Runx1, –2 and –3 genes, whereas only two Runt genes (MgRunxA and MgRunxB) were identified in hagfish (as a representative of jawless vertebrates). The phylogenetic tree (Figure 1) identifies the MgRunxA and MgRunxB genes as being closely related to the Runx1–3 genes. However, it is unknown if these evolved by a hagfish specific duplication or by a Runt gene duplication in the stem species of vertebrates. The phylogenetic analysis of the divergent Runt genes does not give satisfactory high support and a comparative analysis of the Runt gene loci will be needed to resolve this problem. In the pufferfish (T. rubripes) genome, an enigmatic fourth Runt domain gene (FrRunt) was detected in addition to the orthologs of the Runx1, –2 and –3 genes, which appeared to represent either a pufferfish-specific fast evolving derivative of Runx2 or a direct descendant of the ancestral chordate Runt gene [22]. According to our data it is unlikely that the FrRunt gene represents a direct descendent of the ancestral chordate Runt gene which evolved in parallel with the vertebrate Runt genes [22] since we did not detect a FrRunt orthologous gene in tunicates, lancelets, hagfish and dogfish. Instead our phylogenetic analysis (Figure 1) and a comparison of the genomic environment of the FrRunt locus with the genomes of other bony fish (supporting information Figure S2) Figure 7. Runt gene expression in lancelet larvae (B. floridae). Anterior site is located to the left and the dorsal site towards the top. Whole mount in situ hybridization at stages of 16 h (A, B), 26 h (C, D) and 33 h (E, F). A), C) and E) Runt gene exon 1 variant. B), D) and F) Runt gene exon 2 variant. Note that the primary pigment spot, indicated by an arrow, lays in the nerve chord and does not represent a Runt expression domain. An: Anterior notochord, Nt: Neural tube, Nc: Notochord, Hg: Hindgut, Pp: Primary pigment spot, Ppi: Preoral pit. doi:10.1371/journal.pgen.1000025.g007 PLoS Genetics | www.plosgenetics.org 7 March 2008 | Volume 4 | Issue 3 | e1000025