A New Mechanistic Scenario for the Origin and Evolution of Vertebrate Cartilage Maria Cattell1, Su Lai1, Robert Cerny2, Daniel Meulemans Medeiros1* 1 Department of Ecology and Evolutionary Biology, University of Colorado, Boulder, Colorado, United States of America, 2 Department of Zoology, Charles University in Prague, Prague, Czech Republic Abstract The appearance of cellular cartilage was a defining event in vertebrate evolution because it made possible the physical expansion of the vertebrate ‘‘new head’’. Despite its central role in vertebrate evolution, the origin of cellular cartilage has been difficult to understand. This is largely due to a lack of informative evolutionary intermediates linking vertebrate cellular cartilage to the acellular cartilage of invertebrate chordates. The basal jawless vertebrate, lamprey, has long been considered key to understanding the evolution of vertebrate cartilage. However, histological analyses of the lamprey head skeleton suggest it is composed of modern cellular cartilage and a putatively unrelated connective tissue called mucocartilage, with no obvious transitional tissue. Here we take a molecular approach to better understand the evolutionary relationships between lamprey cellular cartilage, gnathostome cellular cartilage, and lamprey mucocartilage. We find that despite overt histological similarity, lamprey and gnathostome cellular cartilage utilize divergent gene regulatory networks (GRNs). While the gnathostome cellular cartilage GRN broadly incorporates Runx, Barx, and Alx transcription factors, lamprey cellular cartilage does not express Runx or Barx, and only deploys Alx genes in certain regions. Furthermore, we find that lamprey mucocartilage, despite its distinctive mesenchymal morphology, deploys every component of the gnathostome cartilage GRN, albeit in different domains. Based on these findings, and previous work, we propose a stepwise model for the evolution of vertebrate cellular cartilage in which the appearance of a generic neural crest-derived skeletal tissue was followed by a phase of skeletal tissue diversification in early agnathans. In the gnathostome lineage, a single type of rigid cellular cartilage became dominant, replacing other skeletal tissues and evolving via gene cooption to become the definitive cellular cartilage of modern jawed vertebrates. Citation: Cattell M, Lai S, Cerny R, Medeiros DM (2011) A New Mechanistic Scenario for the Origin and Evolution of Vertebrate Cartilage. PLoS ONE 6(7): e22474. doi:10.1371/journal.pone.0022474 Editor: Vincent Laudet, Ecole Normale Supe´rieure de Lyon, France Received February 2, 2011; Accepted June 28, 2011; Published July 22, 2011 Copyright: 2011 Cattell et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: RC was supported by the Grant Agency of the Academy of Sciences of the Czech Republic (206/07/P257) and by the Ministry of Education Youth and Sports (project 0021620828). MVC and DMM were supported by National Science Foundation grant IOS0920751 to DMM. SL was supported by an Undergraduate Research Opportunities Program Assistantship from the University of Colorado. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: daniel.medeiros@colorado.edu Introduction A defining feature of the craniate subphylum (vertebrates and hagfish) is the ‘‘New Head’’, which has been linked to the evolution of active predation in the vertebrate lineage[1]. The vertebrate head is a composite structure consisting of paired sense organs, an expanded central nervous system, peripheral ganglia, and a muscular, pumping pharynx. These components are physically supported and protected by cellular cartilage, which is replaced by bone in osteichthian vertebrates. While it can be considered a vertebrate-specific morphological unit, most components of the vertebrate head have clear invertebrate antecedents. For instance, the basic organization of the vertebrate brain has deep roots in the neural tube of invertebrate chordates [2,3], while the paired sense organs and cranial ganglia appear to be derived from evolutionarily ancient cranial placodes and sensory cells[4,5,6]. Similarly, the vertebrate pharynx, though modified for pumping water over the gills and capturing prey, retains the basic respiratory and feeding functions of the pharynx in all deuterostomes. Although much of the vertebrate head likely evolved via the reorganization and augmentation of simpler precursors, the origin of cellular cartilage is less clear. Invertebrates appear to lack any tissue displaying the combination of morphological and biochem- ical properties that defines vertebrate cellular cartilage. In addition, comprehensive analysis of gene expression in amphioxus, a basal chordate, suggests that no single invertebrate cell type coexpresses all, or most, of the genes needed to drive cellular cartilage formation[7]. Rather, individual components of the vertebrate cartilage gene regulatory network (GRN) are expressed in different tissues, most of which are mesoderm-derived. On the other side of the invertebrate/vertebrate transition, all extant vertebrates appear to possess bona fide cellular cartilage, with no tissue constituting an obvious evolutionary intermediate. Consistent with this, work from mouse, zebrafish, and frog suggests that vertebrate cellular cartilage development is mediated by a tightly conserved GRN[8,9,10,11,12,13,14,15,16]. In the head, the vertebrate cartilage GRN is initiated in migrating cranial neural crest cells (CNCC) by the transcription factors SoxE, SoxD, Twist, tfap2, Ets, and Id. After migration, CNCCs activate markers of nascent chondrocytes, including Barx[9,17], Runx[10,18,19, 20,21], and Alx/Cart1 [22,23,24,25]transcription factors. Barx and Runx then work with SoxE and SoxD to drive cartilage PLoS ONE | www.plosone.org 1 July 2011 | Volume 6 | Issue 7 | e22474