regulated by both Sox9 and Barx genes and are downstream of these factors in the cartilage GRN[9,64]. We performed an exhaustive search of P. marinus pre-assembly genome and identified several contigs with similarity to the four most highly conserved deuterostome Runx exons. For three of these exons, we found two distinct aligning lamprey sequences encoding different proteins, suggesting the presence of two lamprey Runx genes (Figure S1, S2). While it is possible that the preassembly P. marinus genome does not include all Runx genes, the level of coverage, and the presence of two Runx genes in hagfish, a related agnathan, suggest this is the full complement of lamprey Runx genes. These genes were designated RunxA and RunxB. RunxA expression was first observed at Tahara st. 25 in the presumptive cranial ganglia and mesenchyme flanking the mouth (Figure 2A). By st. 26.5, the onset of chondrogenesis, expression was seen in the pharyngeal endoderm, cranial ganglia, lateral oral mesenchyme, and intermediate first arch (Figure 2B,E,F). Spots of expression were also observed in the mesodermal core of the pharyngeal arches (Figure 2E). Expression in the cranial ganglia, upper lip, first arch, pharyngeal endoderm and pharyngeal mesoderm was still detectable at st. 27, when cartilage is histologically identifiable by alcian blue staining (Figure 2C,G). By st. 30, when most of the branchial basket cartilage has differentiated, RunxA was still detectable in the pharyngeal endoderm and cranial ganglia (Figure 2D,H). RunxB deployment was much less extensive at early larval stages, marking bilateral spots in the anterior neural tube and cranial ganglia at st. 25 and 26.5 (Figure 2I,J). At st. 27 additional expression was observed in the dorsal fin mesenchyme, around the heart, and in the endostyle (Figure 2K). At st. 30 strong expression was observed in the dorsal fin and around the heart, with reduced expression in the brain (Figure 2L). By 35 d.p.f. weak expression of both RunxA an RunxB was seen in the pharyngeal endoderm. (Figure S3). We then examined Runx, SoxE1, and Col2a1a expression in pre- metamorphic ammocoete larvae and recently metamorphosed juvenile lampreys, to determine if these genes are activated in Vertebrate Cartilage Evolution cellular cartilage around the time of metamorphosis. While we did see expression of Col2a1a in the branchial bars (Figure 3B), we observed no specific signal in any pharyngeal tissue at these stages with either the RunxA (not shown) or RunxB riboprobes (Figure 3G). Similarly, SoxE1 expression above background levels was not seen in pre- or post-metamorphic branchial basket cartilage, though specific expression was seen in the gills, notochord, and a subset of cells in the spinal cord (Figure 3C,H). Taken together, we detected no lamprey RunxA or RunxB expression in branchial basket cartilage at any stage, either before or after metamorphosis. While it is formally possible that an unidentified lamprey Runx paralog is expressed in lamprey branchial basket cartilage, we view this as unlikely. The apparent lack of Runx expression in lamprey cartilage contrasts with gnathostomes where Sox9 and all three Runx genes are broadly co-expressed and interact extensively to regulate chondrogenesis. Assuming RunxA and RunxB represent the full complement of lamprey Runx genes, this difference implies either that Runx lost its ancestral function in cartilage development in the lamprey lineage, or that Runx genes were not part of the ancestral vertebrate cartilage GRN. Recent work using qPCR on adult tissue suggests enrichment of Runx transcripts in the cartilage of adult hagfish, leading to speculation that Runx genes drive skeletogenesis in these agnathans[65]. Hagfish have historically been considered basal jawless craniates, but modern molecular phylogenies support their grouping with lampreys in a single agnathan clade[66]. If Runx is in fact required for chondrogenesis in hagfish, it would support the lineage-specific loss of Runx from the lamprey cartilage GRN. Alternately, Runx expression in adult hagfish could reflect a function in adult tissue maintenance or metabolism unrelated to cartilage specification during development. In jawed vertebrates, Runx genes perform a wide range of functions in embryonic and adult tissues. Gnathostome Runx genes are downregulated in differentiated adult cartilage and upregulated in a variety of other tissues including skin, liver, intestine, thyroid and blood where they Figure 2. Expression of RunxA and RunxB in lamprey larvae. A) Localized expression of RunxA at st. 25 in oral mesenchyme and nascent cranial ganglia. B, C) Expression of RunxA in cranial ganglia (arrow), the intermediate first arch mesenchyme (arrowhead), pharyngeal endoderm, pharyngeal mesoderm, and oral mesenchyme at st. 26.5 and st. 27. D) Expression of RunxA in pharyngeal endoderm at st. 30. E) Section at the level of e in B showing RunxA expression in the pharyngeal pouch endoderm (arrowhead), pharyngeal mesoderm (arrow), and intermediate first arch mesenchyme (black arrowhead). F) Ventral view of B showing strong expression in the mesenchyme flanking the mouth. G) Ventral view of C. H) Ventral view of D. I) A spot of RunxB expression is seen in the nascent cranial ganglia at st. 25. J,K) Expression of RunxB in the brain and cranial ganglia at st. 26.5 and st. 27. At st. 27, weak expression is also visible around the heart, in the endostyle (arrowhead), and in the dorsal fin. L) At st. 30, strong expression of RunxB is seen in the dorsal fin mesenchyme and around the heart, with weaker staining in the brain and pharyngeal endoderm. doi:10.1371/journal.pone.0022474.g002 PLoS ONE | www.plosone.org 4 July 2011 | Volume 6 | Issue 7 | e22474