Figure 4. Expression of Barx in lamprey larvae. A) Barx transcripts at st. 25 in the mesenchyme of the lower lip. B–D) Barx expression at st. 26.5, st. 27, and st. 30 in the lower lip (ventral first arch), intermediate first arch (arrowhead), and in the posterior pharyngeal arches. E) Frontal section at the level of e in B reveals Barx expression in the pharyngeal arches is restricted to the CNCC medial to the pharyngeal mesoderm. Barx transcripts are also detected in the mesenchyme of the lower lip and the intermediate first arch (arrowhead). F–H) Ventral views of B, C, and D, showing expression in the mucocartilage of the lower lip and in the medial aspect of the pharyngeal arches. doi:10.1371/journal.pone.0022474.g004 definitive cellular cartilage of the gill bars. Thus, like Runx, Barx is likely not directly involved in the development of lamprey cellular cartilage, whereas it is required for definitive cartilage formation in gnathostomes. Although Barx expression did not mark CNCC in the lateral pharynx, we did observe Barx expression in CNCC medial to the mesodermal core of each pharyngeal arch. These cells express other markers of CNCC including tfap2, Dlx, and SoxE, as well as fibrillar collagen[39,72]. However, they do not react with alcian blue or correspond to histologically recognizable mucocartilage. In lamprey, this region of the pharyngeal arches gives rise to the smooth muscle of the branchial veins and arteries [43], tissue which is CNCC-derived in gnathostomes. We speculate that lamprey Barx expression in the medial portion of the pharyngeal arches marks CNCC fated to form the branchial vasculature. Lamprey Alx marks upper lip mesenchyme and portions of the branchial basket cartilage Gnathostomes possess three Alx paralogs, Alx3, Alx4, and Cart1. The expression and function of these factors during cartilage development have been studied almost exclusively in mouse, where all three paralogs mark chondrogenic mesenchyme in the frontonasal mass, pharyngeal arches, and limb buds[25,73]. Consistent with their expression patterns, double knock-outs of Alx3 and Alx4 result in severe reductions in the craniofacial and appendicular skeletons[22]. Among invertebrate deuterostomes, Alx is required for skeletal development in sea urchin[74], and amphioxus Alx is expressed in pharyngeal mesoderm thought to secrete acellular cartilage[7], suggesting an ancient role for Alx genes in skeletogenesis. Vertebrate Cartilage Evolution We found multiple contigs corresponding to a single Alx gene in the P. marinus genome (Figure S1, S2), suggesting it is the only lamprey Alx homolog. We then examined its expression in embryos and larvae during cartilage development. Lamprey Alx expression was first seen at st. 23.5 in a spot of mesenchyme in the forming upper lip (data not shown). This expression expanded and intensified to fill the mesenchyme of the upper lip at st. 25 (Figure 5A). Additional expression was seen in the dorsal and ventral aspects of the forming pharyngeal arches and in the forming dorsal fin. By st. 26.5, expression was seen in the mesenchyme of the dorsal and ventral pharyngeal arches, upper lip, and dorsal fin (Figure 5B,F). Expression was excluded from the lower lip mesenchyme and the intermediate portion of the pharyngeal arches. This expression resolved into distinct dorsal and ventral spots in each arch at st. 27 (Figure 5C,G). By st. 30, Alx expression in the pharynx had diminished, but was still detectable in the first arch and subchordal chondrocytes (Figure 5D,H). This expression pattern persisted until 35 d.p.f. (Figure S3), though no Alx expression was detectable in the pharynx of ammocoetes (Figure 3D) or metamorphosed juveniles (not shown). In gnathostomes, SoxE and Alx genes are co-expressed in CNCC- derived chondrogenic mesenchyme[25]. While no regulatory relationship between the two factors has been demonstrated, Sox9 and Alx3/4 double mutant mice have similar hypoplastic skeletal phenotypes, suggesting they operate within the same cartilage Figure 5. Expression of Alx in lamprey larvae. A) Strong Alx expression in the upper lip at st. 25, with weaker expression in the dorsal (arrowhead) and ventral aspects of the forming pharyngeal arches. Expression is also seen in the forming dorsal fin (arrow). B) Expression of Alx in upper lip and strongly in the dorsal and ventral aspects of the posterior (3rd–8th) pharyngeal arches. In the first and second arches, Alx expression is only seen dorsally (arrowheads). Expression is also seen in the dorsal fin (arrow). C) At st. 27, expression in the dorsal and ventral aspects of the pharyngeal arches, upper lip, and dorsal first arch (arrowhead). Expression in the dorsal fin has been lost. D) At st. 30, Alx expression is seen in the dorsal first arch and weakly in the subchordal chondrocytes. E) A frontal section at the level of e in B showing Alx expression in CNCC-derived upper lip mesenchyme surrounding the anteriormost neural tube (arrowhead). Expression is also seen in the dorsal portions of the first arch (arrow) and the posterior arches (double arrowheads). F) Ventral view of B showing expression in the upper lip and the nascent hypobranchial chondrocytes. G) Ventral view of C. H) Ventral view of D. doi:10.1371/journal.pone.0022474.g005 PLoS ONE | www.plosone.org 6 July 2011 | Volume 6 | Issue 7 | e22474