differentiation, partly by activating expression of the structural proteins Col2a1 (fibrillar collagen)[26,27,28] and Aggrecan[29], one of several chondrotin sulfate proteoglycans (CSPGs) expressed in vertebrate cartilage. In the trunk, a similar GRN is activated in mesodermal cells, though its initiation involves the transcription factors Bapx and Pax1/9 [30,31,32]. Conservation of the cartilage GRN in neural crest and trunk mesoderm, and the presence of pharyngeal skeletons in invertebrate deuterostomes and fossil chordates, suggest that cellular cartilage first arose in the pharynx and later expanded into the head and trunk [1,33,34,35,36]. Classical and modern studies suggest that the core features of cellular cartilage development are conserved in the most basal extant vertebrates, the jawless agnathans. The branchial basket cartilage of the agnathan lamprey possesses all of the diagnostic histological and biochemical properties of gnathostome cellular cartilage including stack-of-coins and polygonal morphology, alcian-blue reactivity and fibrillar collagen expression [37,38,39,40,41,42,43,44]. Modern ablation and vital dye labeling show that both gnathostome and lamprey pharyngeal cartilage is derived from cranial neural crest cells (CNCCs)[45,46]. Further- more, lamprey CNCCs coexpress many components of the gnathstome cartilage GRN, including tfap2, Id, Twist, Ets, SoxD, and SoxE[39,47,48,49,50]. While lamprey branchial basket cartilage is likely homologous to definitive gnathostome cellular cartilage, different regions have different properties. The ‘‘hard cartilage’’[42,43,51] in the dorsal portion of the branchial basket skeleton consists of disorganized polygonal chondrocytes and expresses an elastin-like molecule called Lamprin [37,52,53]. In the branchial and hypobranchial bars, discoidal chondrocytes expressing fibrillar collagen and elastin generate so-called ‘‘soft cartilage’’[38,42,43,54]. Lamprey also possesses skeletal tissues with no clear relationship to gnathostome cartilage. Lamprey ‘‘mucocartilage’’ is the main skeletal tissue in the ventral pharynx and oral region[42,43,55]. While biochemically similar to definitive cellular cartilage [42,51], mucocartilage is histologically distinct, consisting of scattered mesenchymal cells embedded in a mucopolysaccharide ma- trix[42,55]. To better understand the evolutionary relationships between agnathan skeletal tissues and gnathostome cartilage, we are analyzing the expression of gnathostome cartilage GRN compo- nents in the sea lamprey Petromyzon marinus. Here we describe the expression of lamprey homologs of three key regulators of gnathostome chondrogenesis, Runx, Barx and Alx/Cart1 from embryonic through larval stages when differentiated cellular cartilage is histologically discernable. Contrary to our expecta- tions, we find that neither Runx nor Barx are co-expressed with SoxE genes in pharyngeal CNCC destined to form the cellular cartilage of the branchial basket. Rather, we see Runx expression in mesenchyme flanking the mouth, and Barx expression in the lower lip and mesenchyme medial to the pharyngeal mesoderm. Similarly, we see restricted expression of Alx in the dorsal and ventral aspects of the branchial basket and in the upper lip, but not in other SoxE and fibrillar collagen-expressing skeletal tissues. Given that Runx, Barx, and Alx are all required for gnathostome cellular cartilage development, our data show that lamprey and gnathostome cellular cartilage develop using divergent GRNs. Furthermore, combinatorial expression of lamprey SoxE, Runx, Alx, and Barx in different populations of presumptive mucocartilage suggest lamprey possess multiple skeletal tissues with genetic features of gnathostome cellular cartilage. Based on these findings, and previous work, we propose a scenario in which early agnathan vertebrates possessed an array of genetically and functionally distinct CNCC-derived skeletal tissues. In the gnathostome Vertebrate Cartilage Evolution lineage, only one of these ancestral tissues was retained and evolved via gene cooption to become the cellular cartilage of modern gnathostomes. Results and Discussion SoxE expression, type A fibrillar collagen expression, and alcian blue staining support common evolutionary and developmental origins for mucocartilage and definitive cellular cartilage In gnathostomes, the SoxE transcription factor, Sox9, is a marker of CNCC and pre-chondrocytes [56]. Gnathostome Col2a1 is a molecular marker for nascent and differentiated cellular cartilage that is directly regulated by Sox9 [26,57,58,59]. Alcian blue binds the chondroitin sulfate proteoglycans (CSPGs, i.e. Aggrecan) secreted by differentiated cellular cartilage, and is considered diagnostic for this tissue [60]. Together, the combined expression of these molecules is used to monitor the development of cellular cartilage in gnathostomes. To obtain a more complete view of lamprey cartilage development, we examined SoxE expression, type A fibrillar collagen expression, and alcian blue reactivity at several time points during chondrogenesis in lamprey (Figure 1). Previous reports have shown strong early expression of the lamprey SoxE paralogs, SoxE1, SoxE2, and SoxE3 in premigratory and early migrating neural crest (Tahara st. 21–23) [61] and expression in the nascent branchial basket (st. 26)[38,39,40,47]. However, expression during chondrogenesis (st. 26.5–30+), and in early post-migratory CNCC (st. 25), has not been reported. We observed a lack of SoxE1 expression at st. 25, and strong expression in the forming branchial bars at st. 26.5, indicating that SoxE1 is temporarily down-regulated sometime between st. 23 and st. 25 and reactivated at st. 26 (Figure 1A–G). At st. 28, expression of SoxE1 in the branchial basket was reduced (Figure 1C,H), particularly in the middle of the vertical branchial bars where cartilage differentiation is first detectable by alcian blue stain- ing[37]. Also at st. 28, we observed broad staining throughout the pharynx including the mesenchyme of the ventral pharynx and oral region. This staining continued until st. 30, when SoxE1 expression in the branchial bar cartilage was undetectable (Figure 1D). The loss of SoxE1 in the branchial bar chondrocytes at st. 30 was coincident with their differentiation as revealed by alcian blue staining (Figure 1I). At 35 days post fertilization (approximately 13 days after st. 30), alcian blue staining revealed the deposition of CSPGs in the fully differentiated branchial basket as well as in the mucocartilage of the oral region (upper lip, first pharyngeal arch, lower lip) and ventral pharynx (Figure 1E, J). Expression of lamprey fibrillar collagen in the forming branchial basket at select time points (st. 25, st. 26) has been described [38,39,62]. We replicated and expanded upon these studies, looking at Col2a1a expression at key stages before, during, and after chondrogenesis. As previously reported, we observed broad Col2a1a expression in post-migratory cranial neural crest cells and somitic mesoderm at st. 25 (Figure 1K). While SoxE1 expression is temporarily downregulated at this stage, cranial neural crest expression of SoxE1 and SoxE2 is seen earlier at st. 23 and st. 24, consistent with Col2a1 regulation by SoxE in CNCC as seen in gnathostomes. At st. 26.5, strong Col2a1a expression is seen in presumptive mucocartilage in the ventral pharynx, upper and lower lips, first pharyngeal arch, and in dorsal fin mesenchyme (Figure 1L,M). Strong Col2a1a expression is also observed in a horizontal band of cells dorsal to the pharyngeal arches at the position of the nascent subchordal cartilage bars. Negligible expression was seen in the forming branchial bars at this stage. At PLoS ONE | www.plosone.org 2 July 2011 | Volume 6 | Issue 7 | e22474