GRN[22,75]. Unlike their gnathostome counterparts, lamprey SoxE and Alx genes are not broadly co-expressed in nascent cellular cartilage. While lamprey SoxE marks the entire branchial basket cartilage, lamprey Alx is restricted to the dorsal and ventral aspects, and is excluded from the vertical branchial bar cartilage. Recent work has shown that the lamprey branchial basket consists of morphologically and molecularly distinct cell types arranged along its dorso-ventral (DV) axis[37,72]. In the central portion of the pharyngeal gill bars, vertically stacked branchial bar chondrocytes display a highly ordered discoidal ‘‘stack-of-coins’’ morphology. Dorsally, subchordal, parachordal and trabecular chondrocytes are disorganized and polygonal in shape. Ventrally, the horizontal hypobranchial bars have a semi-ordered pseudo-discoidal morphol- ogy. Alx expression in the pharynx corresponds to the forming subchordal and hypobranchial chondrocytes. Both subchordal and hypobranchial chondrocytes likely serve as rigid structural elements, in contrast to the central discoidal chondrocytes, which form flexible bars that bend and recoil during pharyngeal pumping. It is possible that Alx expression in the lamprey branchial basket identifies a particular kind of rigid structural cartilage homologous to the cellular cartilage that comprises the bulk of the gnathostome head skeleton. Similarly, Alx expression in the upper lip mesenchyme suggests Alx may also specify a type of mucocartilage. Historical descriptions classify the skeletal tissue in the upper lip, lower lip, first pharyngeal arch, and ventral pharynx as a single kind of generic mucocarti- lage[42,43,51]. However, at the stages we examined, the skeletal tissue around the larval lamprey mouth appears compact, while the presumptive mucocartilage of the ventral pharynx forms a loose mesenchyme. Alx expression in the upper lip may confer some unique physical properties that distinguish this skeletal tissue from the loose mucocartilage in the ventral pharynx. Like Runx, and Col2a1a, we also observed Alx transcripts in the dorsal fin mesenchyme, a tissue that reacts with alcian blue (Figure 1) and is at least partially neural crest-derived [45]. Coexpression of Runx and Alx is not seen in any skeletal tissue in the head, suggesting lamprey dorsal fin mesenchyme may represent a mucocartilage-like tissue unique to the trunk. Summary of SoxE1, Col2a1a, CSPG, Runx, Barx, and Alx expression in lamprey skeletal tissues In sum, our results show expression of SoxE1, Col2a1a, and staining with alcian blue (a proxy for CSPG expression) in all mucocartilage and cellular cartilage of the lamprey head at early larval stages (st. 26.5–st. 30). During this period, RunxA expression was seen in a small subpopulation of mucocartilage flanking the mouth, while RunxB was observed in dorsal fin mesenchyme. Barx expression was observed in a portion of the mucocartilage in the lower lip/first arch, while Alx expression was detected in upper lip mucocartilage, two subpopulations of cellular cartilage, and dorsal fin mesenchyme. No significant overlap of RunxA, RunxB, Barx or Alx expression was seen cellular cartilage (Figure 6), or any other skeletal tissue except in the intermediate first arch (Barx and Runx) the dorsal fin mesenchyme (RunxB and Alx) (Figure 7). However, RunxA, Barx and Alx were all expressed with SoxE1 and Col2a1a in various skeletal tissues and at some point between st. 26.5 and st. 30. In pre-metamorphic ammocoete larvae and post-metamorphic juvenile lampreys, only expression of Col2a1a was detectable above background levels in branchial basket cartilage. A hypothetical scenario for the origin and evolution of vertebrate cartilage We are taking a molecular approach to examine possible evolutionary relationships between invertebrate skeletal tissue, Vertebrate Cartilage Evolution Figure 6. Comparison of lamprey SoxE1, RunxA, Barx, and Alx expression in the pharyngeal arches at st. 26.5. A–D)SoxE1, RunxA, Barx, and Alx expression at st. 26.5 in wholemount in situ- hybridized larvae. While the gnathostome homologs of SoxE1, RunxA, Barx and Alx are all coexpressed in developing cellular cartilage, these genes have largely non-overlapping expression patterns in lamprey. Of the 4 genes, only SoxE1 marks the stack-of-coins cartilage of the vertical branchial bars. Alx marks the subchordal and hypobranchial chondro- cytes as well as the mucocartilage of the upper lip. Barx and Runx are not expressed in lamprey cellular cartilage, but mark different populations of mucocartilage around the mouth. E–G) Expression of SoxE1, RunxA, and Barx in the pharyngeal arches at st. 26.5 as revealed by frontal sections through the middle of the branchial bars. E) Only SoxE1 marks the nascent branchial bar cartilage. F) RunxA is expressed in pharyngeal mesoderm (arrow) and endoderm (arrowhead). G) Barx marks mesenchyme medial to the mesoderm. This mesenchyme also weakly expresses SoxE1 (arrowhead in E). H) Frontal section through h in D showing Alx expression in CNCC cells which will give rise to subchordal chondrocytes. I) Summary of SoxE1, RunxA and Barx expression in the pharyngeal arches at the level of the branchial bars. doi:10.1371/journal.pone.0022474.g006 gnathostome cartilage, and the skeletal tissues of lamprey. Our data show that, despite similarities with gnathostome cellular cartilage, the cellular cartilage of the lamprey branchial basket deploys a rudimentary differentiation program that does not incorporate Runx or Barx. Furthermore, we find that the flexible ‘‘stack of coins’’ chondrocytes of the branchial basket do not express Alx genes and are thus molecularly distinct from the rigid polygonal subchordal and hypobranchial chondrocytes (Figure 6). Finally, we find that lamprey mucocartilages, though morpholog- ically very different from gnathostome cellular cartilage, express various combinations of genes involved in gnathostome chondro- genesis (Figures 6, 7). Taken together, our data reveal an array of histologically and molecularly distinct skeletal tissue types in the larval lamprey head, each displaying a subset of the features seen in gnathostome cellular cartilage (Figure 7). This contrasts with the gnathostome condition in which all embryonic cellular cartilage appears to deploy a single tightly conserved chondrogenic GRN. Assuming PLoS ONE | www.plosone.org 7 July 2011 | Volume 6 | Issue 7 | e22474