Author Summary Important molecular mechanisms underlying mammalian skeletogenesis have been described but knowledge about the evolutionary origin of these gene networks is limited. The Runt gene family (Runx1–3) is of extraordinary impor- tance for skeletogenesis. Runx2 deficient mice completely lack bone. Runx2 and Runx3 are essential for cartilage development and Runx2 regulates the key factor Indian hedgehog, which coordinates skeletogenesis. Here, we reconstructed Runt gene evolution in correlation to skeletal evolution. By analyzing lancelets, one of the closest living relatives of vertebrates, we revealed that the single Runt and Hedgehog family founder genes were co-expressed in primitive skeletal elements of the chordate stem species. Interestingly, at this stage the Runt and Hedgehog pathways were already directly linked to one another. Furthermore we isolated two Runt genes from a representative of jawless cartilaginous fish (hagfish) and three Runt genes from jawed cartilaginous fish (dogfish) which were all expressed in cartilage. The dogfish Runt genes were also found in teeth and placoid scales. This study suggests that Runt genes were involved in all ancient processes of chordate skeletogenesis. Furthermore the analysis supports the theory that most likely the gut was the tissue that originally secreted an acellular gill gut skeleton in the chordate ancestor. Desert hedgehog) also Sonic hedgehog (Shh) signaling is influenced by Runx2 during tooth morphogenesis [11]. Runx2 haploinsufficiency causes the human bone disease cleidocranial dysplasia, further substantiating its importance for skeletal development [12]. Importantly, all three mammalian Runt genes are expressed in cartilage and have been shown to play a role in the formation and differentiation of skeletal elements [6,10,13]. Furthermore, all Runt genes in the mouse are involved in tooth formation [14]. In contrast to the extensively studied Hox genes, which are important for patterning, Runt genes are essential for features that represent evolutionary innovations of vertebrates such as bone [1]. Such innovations result from tinkering with existing processes, from the flexibility that arises from modifications to existing gene networks, and from selective advantage provided by gene duplica- tions or modifications [15]. As simply as this theory explains an important evolutionary process, as difficult it is to functionally analyze how the genetic networks underlying innovations like the vertebrate skeleton evolved. Based on the central role of Runt genes for skeletogenesis in higher vertebrates we hypothesized that these genes played a role in the evolution of cartilage, bone and teeth and thus might be instrumental to understand skeletal evolution in chordates. We therefore analyzed number and expression of Runt genes in hagfish (Myxine glutinosa) as a representative of jawless vertebrates, in dogfish (Scyliorhinus canicula) as a representative of cartilaginous fish and lancelets (Branchiostoma lanceolatum and B. floridae) as representatives of celphalochordates to reconstruct if Runt genes were already expressed in the developing skeleton of the chordate, vertebrate and jawed vertebrate stem species. In addition, we tested if Runt and Hh are co-expressed in lancelets and if a functional interaction between the Runt and Hh pathways might have evolved before the cellular cartilage of vertebrates evolved. In this study we show that the stem species of chordates harboured a single Runt gene, whereas three Runt genes were present before the emergence of gnathostomes. Runt genes are expressed in developing cartilage, teeth and placoid scales of cartilaginous fish and cartilage of jawless vertebrates. In adult lancelets the Runt gene is expressed together with Hh, in the endo- and ectoderm of the gill Runt Gene Evolution bars. Furthermore, we demonstrate that the lancelet Runt protein can directly bind to and activate the lancelet Hh promoter. This suggests that beside SoxE and fibrillar collagen two other key factors for vertebrate skeletogenesis (Runt and Hh) were part of an ancient gene network for skeletogenesis in the gill gut stabilizing the gill bars of the common ancestor of vertebrates and lancelets approximately 700 million years ago. Our finding that the gut is an ancient Runt expression domain of deuterostomes is in accordance with the hypothesis that endodermal secretion was the ancestral mode of making pharyngeal cartilage [5]. Results Isolation of hagfish and dogfish Runt genes We used a PCR-based approach using cDNA as well as genomic DNA to identify Runt genes in lower vertebrates. This led to the detection of two Runt genes in hagfish (MgRunxA, MgRunxB) and three Runt genes in dogfish (ScRunx1–3). All of these newly detected Runt genes had a 39 end with the characteristic VWRPY-motif. The two different 59 ends of the Runt genes amplified from embryonal dogfish cDNA were homologous to the 59 mammalian promoter variant-1 (MASNS-like) and variant-2 (MRIPV-like) motifs, respec- tively. In the two hagfish Runt genes amplified from adult hagfish cDNA only a single 59 gene end was detected. According to our Blast searches against the Ensembl pre-genome sequences of lamprey (Pteromyzon marinus) the two hagfish 59 ends represent most likely the promoter variant 2. Because of the unavailability of hagfish embryos it could not be clarified if two Runt gene promoter 1 variants are expressed during early hagfish development. Blast searches in whole genome databases (NCBI, JGI, Ensembl) revealed that there are most likely two Runt genes in the lamprey genome, and one Runt gene in cnidarians (Nematostella vectensis), nematodes (Caenorhabditis elegans), cephalochordates (B. floridae), and tunicates (Ciona intestinalis, Oikopleura dioica) [16,17]. We detected two Runt genes in sea urchin (Strongylocentrotus purpuratus) [18,19], which were located on the same genomic contig, two partial Runt genes in skate (Raja eglanteria) [20], three Runt genes in mammals [6,7] and four in pufferfish (Takifugu rubripes) [21,22] and also four in zebrafish (Danio rerio) including a duplicated Runx2 gene [23]. In chicken (Gallus gallus) three Runt genes were found. An alignment of all newly detected Runt genes together with other deuterostome Runt genes is provided as supporting information (Figure S1) and the GeneBank accession numbers are given in the footnote. Conserved synteny of Runt and the chloride intracellular channel (Clic) genes in human, chicken and tunicate genomes Comparable to the human Runt loci [24], the three orthologous chicken Runt genes are followed by a Clic gene on the complementary strand. The chicken Runx1 on chromosome 1 is followed by a Clic6 homologous gene, the chicken Runx2 on chromosome 3 by a Clic5 homologous gene and the chicken Runx3 on chromosome 23 is followed by a Clic4 homologous gene. In lancelet the Runt and Clic genes are located on different scaffolds (JGI assembly vers 1.0). However, in the genome of the tunicate C. intestinalis a Clic homologous gene was found in proximity to Runt on chr_12q (JGI, Assembly vers 2.0). This strongly suggests that the entire Runt locus was triplicated during the evolution of chordates. The last common ancestor of chordates harboured a single Runt gene Our phylogenetic analysis (Figure 1) suggests that the stem species of chordates harboured a single Runt gene, whereas the last PLoS Genetics | www.plosgenetics.org 2 March 2008 | Volume 4 | Issue 3 | e1000025