© 2014. Published by The Company of Biologists Ltd | Development (2014) 141, 629-638 doi:10.1242/dev.097261 629 ABSTRACT A defining feature of vertebrates (craniates) is a pronounced head supported and protected by a cellularized endoskeleton. In jawed vertebrates (gnathostomes), the head skeleton is made of rigid three- dimensional elements connected by joints. By contrast, the head skeleton of modern jawless vertebrates (agnathans) consists of thin rods of flexible cellular cartilage, a condition thought to reflect the ancestral vertebrate state. To better understand the origin and evolution of the gnathostome head skeleton, we have been analyzing head skeleton development in the agnathan, lamprey. The fibroblast growth factors FGF3 and FGF8 have various roles during head development in jawed vertebrates, including pharyngeal pouch morphogenesis, patterning of the oral skeleton and chondrogenesis. We isolated lamprey homologs of FGF3, FGF8 and FGF receptors and asked whether these functions are ancestral features of vertebrate development or gnathostome novelties. Using gene expression and pharmacological agents, we found that proper formation of the lamprey head skeleton requires two phases of FGF signaling: an early phase during which FGFs drive pharyngeal pouch formation, and a later phase when they directly regulate skeletal differentiation and patterning. In the context of gene expression and functional studies in gnathostomes, our results suggest that these roles for FGFs arose in the first vertebrates and that the evolution of the jaw and gnathostome cellular cartilage was driven by changes developmentally downstream from pharyngeal FGF signaling. KEY WORDS: Lamprey, FGF, Pharynx, Cartilage, Vertebrate evolution INTRODUCTION Based on the fossil record and the morphology of living chordates, it has been proposed that the head skeleton of the first vertebrates consisted of thin bars of flexible cellular cartilage (Mallatt and Chen, 2003; Shu et al., 2003). This primitive skeleton provided elastic recoil for pharyngeal pumping, which facilitated gill ventilation and filter feeding (Gans and Northcutt, 1983). In the gnathostome (jawed vertebrate) lineage, both the composition and organization of the vertebrate pharyngeal skeleton were modified. The simple bars supporting each pharyngeal arch were replaced by multiple RESEARCH ARTICLE 1 Department of Ecology and Evolutionary Biology, University of Colorado, Boulder, Boulder, CO 80309, USA. 2 Department of Zoology, Comenius University in Bratislava, 84215 Bratislavia,, Slovakia. 3 Department of Zoology, Charles University in Prague, 116 36 Prague, Czech Republic. *Present address: Department of Organismic and Evolutionary Biology, Harvard University, Cambridge, MA 02138, USA. ‡ Author for correspondence (daniel.medeiros@colorado.edu) Received 6 April 2013; Accepted 8 November 2013 articulating elements with complex three-dimensional morphologies. Concurrently, flexible elastin-rich cellular cartilage was replaced with a mixture of rigid collagen-rich cartilage and intervening soft joint tissue. Precisely how these changes occurred is unknown, but they presumably involved alterations to both head skeleton patterning and the genetic program underlying cellular cartilage differentiation. In jawed vertebrates, the fibroblast growth factors FGF3 and FGF8 are expressed in pharyngeal endoderm and ectoderm, and their loss leads to defects in both head skeleton organization and cartilage differentiation. In zebrafish and mouse, these defects are accompanied by disruptions in pharyngeal pouch formation, suggesting that FGFs help pattern the head skeleton in part by mediating pharyngeal segmentation (Abu-Issa et al., 2002; Crump et al., 2004; Walshe and Mason, 2003). Evidence also indicates that FGF signals act directly on skeletogenic cranial neural crest cells (NCCs) to regulate head skeleton development. In zebrafish, chick and mouse, FGF receptors (FGFRs) are expressed by cranial NCCs (Abu-Issa et al., 2002; Sarkar et al., 2001; Yamauchi et al., 2011), and exposure of chick mandibular mesenchyme to ectopic FGFs inhibits expression of Bapx and formation of the jaw joint (Wilson and Tucker, 2004). FGFs also perform a patterning function in mouse, in which different levels of FGF8 signaling modulate gene expression and odontogenic potential along the rostral-caudal axis in mandibular arch NCCs (Tucker et al., 1999). In zebrafish, evidence supports a more general role for FGFs in specifying skeletogenic fate. Expression of dominant-negative FGFRs in zebrafish NCCs causes broad downregulation of the chondrogenic NCC marker dlx2 (Das and Crump, 2012), and treatment with the FGFR inhibitor SU5402 causes loss of barx expression in chondrogenic NCCs and complete failure of chondrogenesis (Sperber and Dawid, 2008; Walshe and Mason, 2003). Similarly, in chick, transfection of mandibular NCCs with dominant-negative FGFRs results in locally reduced Sox9, Runx and Aggrecan expression and hypoplastic cartilage elements (Havens et al., 2008). Furthermore, under cell culture conditions, chick NCC-derived prechondrocytes require FGF signaling for survival, Sox9 expression and overt chondrogenesis, and Fgf8 is sufficient to induce Barx expression (Barlow et al., 1999; Sarkar et al., 2001). To better understand the evolution of the vertebrate head skeleton we, and others, have been investigating skeletogenesis in the jawless vertebrate, lamprey. Lamprey diverged from gnathostomes shortly after vertebrate origins and is the most basal vertebrate readily accessible to embryonic manipulation. Although lamprey possesses a head skeleton incorporating cellular cartilage it is thought to retain the basic structure and function of the head skeleton of early vertebrates. In contrast to the rigid, jointed cartilage elements of gnathostomes, the pharyngeal component of the lamprey head skeleton consists of thin rods of cellular cartilage that secrete an Roles for FGF in lamprey pharyngeal pouch formation and skeletogenesis highlight ancestral functions in the vertebrate head David Jandzik 1,2 , M. Brent Hawkins 1, *, Maria V. Cattell 1 , Robert Cerny 3 , Tyler A. Square 1 and Daniel M. Medeiros 1,‡ Development