Vertebrate Cartilage Evolution Figure 1. Expression of SoxE1 and fibrillar collagen, and alcian blue staining during chondrogenesis in lamprey larvae. A) SoxE1 expression is not observed in post-migratory CNCC at st. 25. B) Strong, specific expression of SoxE1 in the nascent branchial basket cartilage at st. 26.5. C) Expression of SoxE1 in the branchial basket cartilage and broadly in the head mesenchyme at st. 28. Compared to st. 26.5, SoxE1 expression in the branchial bar cartilage is reduced (arrowhead in inset, showing the pharyngeal arch indicated by the asterisk). D) At st. 30, SoxE1 expression is detectable throughout the head mesenchyme, but has been downregulated in the branchial basket cartilage. E) Alcian blue staining at 35 days post- fertilzation (d.p.f.) in the branchial basket and the mucocartilage of the upper lip, oral region, and ventral pharynx as well as the dorsal fin. Strong staining is also seen in the notochord (arrowhead). F) Frontal section at the level of f in B showing SoxE1 expression in the nascent branchial basket cartilage and weakly in the CNCC medial to the pharyngeal mesoderm (arrow). G) Ventral view of B showing expression in the mucocartilage around the mouth. H) Ventral view of C. I) Alcian blue staining at st. 30 in the differentiated branchial basket cartilage, notochord, and weakly in the nascent mucocartilage (arrow). J) Ventral view of E showing alcian blue reactivity in the mucocartilage of the ventral pharynx. K) Expression of the lamprey fibrillar collagen gene Col2a1a at st. 25. Expression is seen in the somites (arrow) and post-migratory neural crest cells in the pharynx (arrowhead). L) Col2a1a expression at st. 26.5 throughout the head mesenchyme including presumptive mucocartilage in the ventral pharynx and around the mouth, and in the presumptive subchordal cartilages(arrow). Minimal expression is seen in the vertical branchial bars. Col2a1a mRNA is also present in the dorsal fin (arrowhead) M) Ventral view of L showing mucocartilage expression. N) Broad expression of Col2a1a throughout the pharynx and oral region including presumptive mucocartilage and branchial basket cartilage at st. 30. O) At 35 d.p.f. Col2a1a is still broadly expressed the head skeleton. doi:10.1371/journal.pone.0022474.g001 st. 28 strong expression of Col2a1a was detected throughout the mucocartilage around the mouth, with weaker expression in branchial basket cartilage (Figure 1N). Similar expression was seen later at 35 d.p.f. (Figure 1O). Our results show SoxE1 expression is high in post-migratory pharyngeal CNCC, where it is co-expressed with Col2a1, and is then downregulated as these cells differentiate into cellular cartilage. This sequence mirrors the down-regulation of Sox9 seen in differentiating gnathostome cartilages and supports conserved roles for SoxE in lamprey branchial basket cartilage and gnathostome cellular cartilage development as previously proposed [40,47]. In addition to the branchial basket, however, we also observed SoxE1 expression and alcian blue staining throughout the developing mucocartilage of the oral apparatus and ventral pharynx. Furthermore, we found that these tissues express high levels of fibrillar collagen [38,39] as originally suggested by Schaffer[51] and Johnels[42]. Due to its staining properties and mesenchymal morphology, mucocartilage is generally considered a derived form of connective tissue unrelated to cellular cartilage[55]. However, co-expression of fibrillar collagen, SoxE, and CSPGs in mucocartilage suggest some developmental and/or evolutionary relationship between mucocartilage and cellular cartilage. Hardisty[44] proposed that mucocartilage may represent an undifferentiated embryonic tissue that is carried over into the larval stage. An alternate hypothesis is that mucocartilage represents an evolutionary precursor to bona fide cellular cartilage. We decided to further investigate these possibilities by looking at the expression of three other key regulators of vertebrate chondrogenesis; Runx, Barx, and Alx/Cart1, in the lamprey head skeleton. Runx genes are expressed in lamprey mucocartilage, but not in the branchial basket cartilage Runx genes are key regulators of both cartilage and bone development in jawed vertebrates. During embryogenesis all three gnathostome Runx paralogs, Runx1,2,3[18] are expressed in presumptive chondrocytes, and Runx2 and Runx3 have been shown to be required for their differentiation into cellular cartilage[10,20,21]. After chondrogenesis, Runx genes regulate the formation of replacement cartilage and the ossification of endochondral bone[63]. Functional studies suggest Runx genes are PLoS ONE | www.plosone.org 3 July 2011 | Volume 6 | Issue 7 | e22474