diversity was coincident with the consolidation of several rudimentary skeletogenic GRNs into a single chondrogenic GRN. Thus, during gnathostome evolution, gene programs operating primitively in different types of skeletal tissue became co-expressed in a single type of cellular cartilage. We previously proposed that genetic cooption of mesodermal gene networks by the evolving cranial neural crest drove the origin of vertebrate cellular cartilage[7]. Our current results suggest similar cooption events occurred during the evolution of gnathostome cellular cartilage. Below we integrate our current and previous data and propose a stepwise mechanistic scenario for the origin and evolution of vertebrate cellular cartilage (Figure 8). Step 1: The origin of skeletogenic CNCC Our current results, and previous work, reveal a set of core features shared by all skeletogenic neural crest cells, including gnathostome cellular cartilage, lamprey cellular cartilage, and lamprey mucocartilage. These include; 1) differentiation from a mesenchymal precursor cell, 2) persistent expression of Twist, Ets, Id, and SoxE after migration into the pharynx[39,47], 3) secretion of CSPGs[37], and 4) expression of fibrillar collagen. Notably, this list does not include expression of Runx or Barx, which our data show are not general markers of neural crest-derived skeletal tissue. Using these criteria as a minimal definition of vertebrate neural crest-derived skeletal tissue, we can then ask if any protochordate tissue possesses all, or most, of these features. As has been shown previously, Twist, Ets, Id, Alx, and fibrillar collagen are coexpressed in the pharyngeal mesoderm of amphioxus larvae[7,50]. Further- more, this tissue displays mesenchyme-like cell morphology and may give rise to acellular cartilage bars composed of CSPGs[7,50]. Thus, skeletogenic mesoderm with most attributes of skeletogenic neural crest cells, but lacking SoxE expression, was likely present in the first chordates. The existence of migratory neuroblasts in a range of invertebrates [76,77,78] suggests that the vertebrate ancestor also possessed some kind of migratory neural tube cell. Assuming that co-expression of Twist, Ets, Id, Alx, and fibrillar collagen reflects a conserved GRN, it is possible that skeletogenic neural crest cells evolved via activation of this GRN in non-skeletogenic migratory neural tube cells. The lack of SoxE expression in amphioxus pharyngeal mesoderm further suggests this cooption involved the integration of SoxE into the evolving skeletogenic GRN. How these changes occurred is unclear. Aside from their function in skeletogenesis, vertebrate SoxE paralogs are required for the specification and differentiation of all neural crest cell lineages, as well as for the development of glia and the otic placode[79]. In addition, amphioxus SoxE is expressed throughout the nascent nervous system in early larvae, but not in any obviously skeletogenic tissues aside from a few cells in the notochord [80]. These data imply that the ancestral function of chordate SoxE was in neural development, rather than skeletogenesis, as has been proposed [65,81]. Furthermore, these results suggest that migra- tory neural tube cells could have acquired SoxE simply by maintaining SoxE expression after leaving the CNS. Based on these observations, we propose that skeletogenic neural crest cells evolved by activation of a mesodermal skeletogenic GRN in migratory neural tube cells expressing SoxE. (Figure 8A,B). A role for SoxE genes in skeletogenesis then evolved later in the vertebrate lineage as SoxE was integrated into this skeletogenic GRN. The cartilage GRN in vertebrates is induced and maintained by signals secreted by the pharyngeal endoderm and ectoderm, including Hedgehogs and FGFs[82,83]. Provocatively, FGF and Hedgehog ligands are expressed in the amphioxus pharynx at larval Vertebrate Cartilage Evolution stages when skeletogenesis is likely initiated[7,84]. We further speculate that the activation of mesodermal skeletogenic gene programs could have evolved as proto-CNCC cells gained responsiveness to these signals. Based on Runx and SoxE co-expression in the foregut of adult amphioxus, it has been proposed that cooption of a endodermal skeletogenic GRN(s) incorporating these factors drove the appearance of skeletogenic CNCC[65,81]. However, expression of lamprey SoxE1 and Runx in non-skeletogenic pharyngeal endoderm and gills (Figure 2, Figure 3B) suggests these factors perform non-skeletogenic functions in chordate endoderm. Furthermore, our data showing that Runx is not expressed in lamprey cellular cartilage implies it is not a core feature of chordate skeletal development. Finally, the early role of SoxE genes in the initial formation of all neural crest cells, suggests its ancestral function was in neural crest cell specification rather than chondrogenesis[79]. Thus, cooption of Runx and SoxE from endoderm was likely not involved in the origin skeletogenic neural crest cells. Instead, our data support later recruitment of Runx by SoxE-expressing skeletogenic neural crest cells in the gnathostome lineage (see below). Step 2: The diversification of CNCC-derived skeletal tissues in early jawless vertebrates The ‘‘New Head’’ hypothesis suggests that the original function of vertebrate cartilage was to provide structural support for pharyngeal pumping[1]. The concurrent expansion of the CNS, anterior sense organs, and cranial ganglia also likely required additional skeletal support, as did the novel structural demands on the oral region of early vertebrate predators. In the first vertebrates, we posit that these various functions were performed by a generic CNCC-derived skeletal tissue expressing Twist, Id, Ets, Alx, SoxE, and fibrillar collagen, and secreting CSPGs (Figure 8B). Based on gene expression in lamprey, we further speculate that this generic CNCC-derived head skeleton then diversified, with different regions taking on specialized properties in response to particular structural demands. During this phase of vertebrate skeletal evolution, Runx, and Barx, were recruited to different parts of the CNCC-derived skeleton, and Alx, which was initially expressed in all skeletogenic CNCC, was lost from some regions. These changes in gene expression drove the establishment of several different skeletal tissue types with distinct physical properties (Figure 8C). Among these were compact tissues composed of cells with polygonal and discoidal morphologies that would be histologically identifiable as proper ‘‘cellular cartilage’’, and several types of mesenchymal skeletal tissues in the head and dorsal fin. This primitive diversity of CNCC-derived skeletal cell types is thus preserved in lamprey, where Runx, Barx, and Alx genes mark different subsets of cartilage and mucocartilage around the mouth and in the pharynx (Figure 7). Localized expression of these factors would also require some level of underlying anteroposterior (AP) and dorsovental (DV) patterning in the oro-pharyngeal region. Consistent with this, recent work suggests sophisticated gnathostome-like AP and DV patterning systems were in place before the divergence of jawed and jawless vertebrates[72,85]. Step 3: The evolution of gnathostome cartilage While chondrogenic gene expression in lamprey identifies several molecularly distinct skeletal tissues (Figure 7), the ubiquitous coexpression of chrondrogenic GRN components in the gnathostome head skeleton suggests the presence of only one major cartilage type. If lamprey represents the basal state, then the evolution of the gnathostome head skeleton was associated with a reduction in skeletal tissue diversity. A hallmark of the lamprey PLoS ONE | www.plosone.org 9 July 2011 | Volume 6 | Issue 7 | e22474