Figure 3. Expression of cartilage GRN components in pre- metamorphic ammocoete larvae and post-metamorphic juve- nile lamprey. A) Sections for the in situ hybridizations shown in B–E were cut from pre-metamorphic ammocoete larvae through the pharyngeal arches at approximately the level of the dotted line. B) Strong expression of Col2a1a is seen above background levels in the notochord (arrow) and in cells in and around the branchial bar cartilage (arrowhead). C) Specific expression of SoxE1 in the gills (arrowhead). Diffuse staining in the branchial cartilage (arrowhead) is likely due to background as can be seen with two negative control riboprobes corresponding to a lamprey SoxB1 gene (I, arrowhead) and Xenopus laevis FGF3 (J, arrowhead). No specific expression of Alx (D) or Barx (E) was seen in branchial bar cartilages. F) Sections for the in situ hybridizations shown in G–J were cut from post-metamorphic juvenile lampreys through the pharyngeal arches at approximately the level of the dotted line. No expression of RunxB (G) or SoxE1 (H) was seen in the branchial basket cartilage (arrowheads) after metamorphosis. In contrast, strong, specific reactivity with the SoxE1 and SoxB1a probes is seen in different subsets of spinal cord neurons (H, I, insets). SoxE1 mRNA is also detected in the notochord (H, arrow), and gills (H, double arrowhead). I) Control in situ hybridization with lamprey SoxB1a showing neural expression, and expression in the gills. doi:10.1371/journal.pone.0022474.g003 regulate cell division and stem cell quiescence [19,67,68,69]. Consistent with a general role in adult tissue maintenance, hagfish Runx transcripts were found in every hagfish tissue examined, with slightly higher levels in hard cartilage[65]. In either case, the lack of detectable enrichment of lamprey Runx mRNA in branchial basket cartilage indicates Runx genes are dispensable for the development of histologically discernable cellular cartilage. We propose this reflects the basal vertebrate state, but it could formally Vertebrate Cartilage Evolution represent the presence of a derived chondrogenic GRN specific to lampreys. Runx expression during cartilage development in hagfish embryos would help distinguish between these two scenarios. While lamprey Runx expression was not observed in the definitive cellular cartilage of the branchial basket, strong, localized RunxA expression was observed in subpopulations of nascent mucocartilage around the mouth and in the intermediate portion of the first arch. This restricted expression suggests lamprey Runx genes may be performing tissue-specific functions in the development of particular kinds of mucocartilage, rather than acting as general drivers of skeletogenesis as they are in gnathostomes. Other domains of Runx expression included the brain, cranial ganglia, and pharyngeal endoderm in early larvae (st. 25–26.5) and the heart, and dorsal fin mesenchyme in late larvae (st. 27–30). Expression of Runx genes is observed in the cranial ganglia of all gnathostomes examined, indicating a deeply conserved role for Runx in the development of these structures[18,21]. Expression in the dorsal fin may be related to a skeletogenic function as these cells also express SoxD and fibrillar collagen (Figure 1)[38], and react with with alcian blue (Figure 1E). An endodermal specification function for Runx genes appears to be evolutionarily ancient as gnathostomes, amphioxus, sea urchin, and C. elegans all express Runx in the nascent gut, and Runx is required for gut formation in C. elegans [7,67,70]. Runx expression in the pharyngeal endoderm of adult amphioxus has been interpreted as evidence of a rudimentary skeletogenic program in this tissue[65]. Given the pleiotropic nature of Runx function, and the pan-metazoan expression of Runx genes in endoderm, it seems equally likely that Runx genes are performing some other function in this tissue. Lamprey Barx is expressed in lower lip mucocartilage and CNCC in the medial pharyngeal arches, but not in developing branchial bar cartilage Recent work has shown that Barx is required for the differentiation of CNCC-derived cartilage in the zebrafish head where it is downstream of FGF signaling and regulates Runx expression[9]. Similar broad expression of Barx in the CNCC of chick and mouse suggest this role is conserved among all gnathostomes[17,71]. In addition, functional studies in mouse have demonstrated an essential role for Barx in the chondrogenesis of the mesoderm-derived appendicular skeleton[27]. We isolated a lamprey Barx ortholog, and described its expression in the first pharyngeal arch at st. 26.5 in a previous study[72]. Here we detail lamprey Barx expression from embryonic through late larval stages in all tissues. Barx transcripts are first weakly detectable in a stream of CNCCs migrating into the region of the first pharyngeal arch at st. 24 (data not shown). At st. 25, Barx expression is seen in the lower lip and the forming pharyngeal arches (Figure 4A). At st. 26.5 and 27, strong Barx expression is seen in the lower lip, extending dorsally into the intermediate region of the first pharyngeal arch as previously described (Figure 4B,F,C,G). Expression is also seen in presump- tive CNCC-derived mesenchyme positioned medial to the pharyngeal arch mesoderm in the posterior arches (Figure 4E). These domains of expression persist into st. 30 when chondro- genesis of the branchial bars is complete (Figure 4D,H). Similar expression is still apparent at 35 d.p.f (Figure S3). No Barx expression was seen in the pharynx of pre-metamorphic ammocoete larvae (Figure 3E) or post-metamorphic juveniles (not shown). As with Runx, we noted an absence of Barx expression in the laterally-positioned pharyngeal CNCCs which give rise to the PLoS ONE | www.plosone.org 5 July 2011 | Volume 6 | Issue 7 | e22474