Vertebrate Cartilage Evolution Figure 8. A hypothetical scenario for the origin and evolution of vertebrate cartilage. A) Based on the expression of SoxE genes throughout the neural tube of amphioxus larvae, and the presence of migratory neural cells in urochordates and other invertebrates, we posit that the ancestral pre-vertebrate chordate possessed migratory neural tube cells that expressed SoxE genes (light blue). These cells migrated into the pharynx and generated neural and/or pigment cells, but were not skeletogenic. The pharyngeal skeleton at this stage (pink) resembled that of amphioxus, consisting of acellular cartilage incorporating acid mucopolysaccharides and secreted by pharyngeal mesoderm expressing Twist, Ets, Id, Alx, CSPGs and fibrillar collagen. B) The origin of bona fide skeletogenic neural crest, and the first craniates, was marked by the activation of this mesodermal skeletogenic gene program in SoxE-expressing migratory neural tube cells (light blue). This ancestral form of cellular cartilage provided structural support for the evolving head. C) In the lineage leading to the common ancestor of modern jawed and jawless vertebrates, this generic skeletal tissue diversified into several subtypes with distinct molecular and structural properties via cooption of Runx and Barx and the selective loss of Alx expression. This included definitive ‘‘cellular cartilage’’ similar to that seen in gnathostomes and multiple mucocartilage subtypes as seen in lamprey. This basal condition is maintained in lamprey. D) In gnathostomes this heterogenous head skeleton was replaced with a skeleton consisting mainly of a single type of rigid cellular cartilage. Coincident with this shift was the consolidation of SoxE1, Runx, Barx, and Alx genes into a single gnathostome cellular cartilage gene network. Based on similar gene expression and cell morphology, the joint tissue seen in gnathostomes (light blue) may represent a basal form of skeletal tissue similar to mucocartilage in the ventral pharynx of lamprey. doi:10.1371/journal.pone.0022474.g008 head skeleton is the preponderance of soft, flexible skeletal tissues such as the mucocartilage of the oral region and ventral pharynx, and the discoidal ‘‘soft’’ cartilage of the branchial basket. This contrasts with the gnathostome condition, where uniformly rigid cartilage elements articulate at foci of soft joint tissue. Rigid ‘‘hard’’ cartilages have been described in lamprey based on their staining properties[42,43,51]. Interestingly, these cartilages have the polygonal morphology typical of most gnathostome cartilages and express Alx. We speculate that a similar type of specialized, polygonal, Alx-positive cartilage was present in the first vertebrates where it constituted a small portion of the head skeleton. In the gnathostome lineage, this rigid cartilage proliferated, becoming the dominant component of the head skeleton (Figure 8D). The broad expression of Runx and Barx in the gnathostome head skeleton suggests this transition also involved the expanded expression of these factors. We posit that Runx and Barx may have initially conferred novel structural properties upon this tissue. The transition to rigid structural cartilage may have been driven by the novel physical requirements of holding and processing increasingly large prey items and supporting and protecting a large CNS and sense organs. Similar selective pressures may have driven the subsequent evolution of bone. In addition to rigid cellular cartilage, gnathostomes also possess soft joint tissue between cartilaginous elements in the head. Like cellular cartilage, these cells are CNCC-derived and initially express SoxE, Twist, Ets, and Id and stain with alcian blue [86]. However, unlike, cellular cartilage, this tissue remains mesenchy- mal and does not express Barx and Runx [9,60]. Based on PLoS ONE | www.plosone.org 10 July 2011 | Volume 6 | Issue 7 | e22474