Evolution of a Core Gene Network for Skeletogenesis in Chordates Jochen Hecht1,2, Sigmar Stricker2., Ulrike Wiecha2., Asita Stiege2., Georgia Panopoulou 2 , Lars Podsiadlowski3, Albert J. Poustka2, Christoph Dieterich4, Siegfried Ehrich5, Julia Suvorova2, Stefan Mundlos1,2,6, Volkhard Seitz2* 1 BCRT, Universita¨tsmedizin Charite´, Berlin, Germany, 2 Max Planck Institute for Molecular Genetics, Berlin, Germany, 3 Department of Animal Systematics and Evolution, Free University, Berlin, Germany, 4 MPI for Developmental Biology Department 4 - Evolutionary Biology, T u¨bingen, Germany, 5 Bundesforschungsanstalt f u¨r Fischerei, Hamburg, Germany, 6 Institute for Medical Genetics, Charite´, Universita¨tsmedizin Berlin, Berlin, Germany Abstract The skeleton is one of the most important features for the reconstruction of vertebrate phylogeny but few data are available to understand its molecular origin. In mammals the Runt genes are central regulators of skeletogenesis. Runx2 was shown to be essential for osteoblast differentiation, tooth development, and bone formation. Both Runx2 and Runx3 are essential for chondrocyte maturation. Furthermore, Runx2 directly regulates Indian hedgehog expression, a master coordinator of skeletal development. To clarify the correlation of Runt gene evolution and the emergence of cartilage and bone in vertebrates, we cloned the Runt genes from hagfish as representative of jawless fish (MgRunxA, MgRunxB) and from dogfish as representative of jawed cartilaginous fish (ScRunx1–3). According to our phylogenetic reconstruction the stem species of chordates harboured a single Runt gene and thereafter Runt locus duplications occurred during early vertebrate evolution. All newly isolated Runt genes were expressed in cartilage according to quantitative PCR. In situ hybridisation confirmed high MgRunxA expression in hard cartilage of hagfish. In dogfish ScRunx2 and ScRunx3 were expressed in embryonal cartilage whereas all three Runt genes were detected in teeth and placoid scales. In cephalochordates (lancelets) Runt, Hedgehog and SoxE were strongly expressed in the gill bars and expression of Runt and Hedgehog was found in endo- as well as ectodermal cells. Furthermore we demonstrate that the lancelet Runt protein binds to Runt binding sites in the lancelet Hedgehog promoter and regulates its activity. Together, these results suggest that Runt and Hedgehog were part of a core gene network for cartilage formation, which was already active in the gill bars of the common ancestor of cephalochordates and vertebrates and diversified after Runt duplications had occurred during vertebrate evolution. The similarities in expression patterns of Runt genes support the view that teeth and placoid scales evolved from a homologous developmental module. Citation: Hecht J, Stricker S, Wiecha U, Stiege A, Panopoulou G, et al (2008) Evolution of a Core Gene Network for Skeletogenesis in Chordates. PLoS Genet 4(3): e1000025. doi:10.1371/journal.pgen.1000025 Editor: Sebastian Shimeld, University of Oxford, United Kingdom Received July 12, 2007; Accepted February 7, 2008; Published March 21, 2008 Funding: No external funding was granted for this study. Competing Interests: The authors have declared that no competing interests exist. * E-mail: seitz_v@molgen.mpg.de . These authors contributed equally to this work. Copyright: 2008 Hecht 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. Introduction The skeleton is a hallmark of vertebrates and has been widely used over the past decades for phylogenetic analyses [1]. However, little is known about its molecular evolution. Descriptive data are available for the matrix proteins produced by the cells that constitute the skeleton in jawless vertebrates (epitomized by hagfish and lampreys, collectively termed agnathans). Beside species specific proteins [2] they possess cartilage with type II collagen (Col2a1), which is also the characteristic matrix protein for jawed vertebrates (gnathostomes) [3,4]. Furthermore Sox9, which directly regulates Col2a1 in mammals, was shown to be expressed in cartilage of the lamprey [3]. Interestingly SoxE (an invertebrate homolog to the mammalian Sox8/9/10) was found to be co-expressed with fibrillar collagen in the hemichordate Saccoglossus bromophenolosus [5]. The expression was found in the pharyngeal endodermal cells, which are most likely responsible for the secretion of an acellular cartilage. Such an endodermal secretion was postulated to be primarily the ancestral mode of making pharyngeal cartilage in deuterostomes [5]. Up to now no Runt gene expression has been described in skeletal elements of lancelets, agnathans and jawed cartilaginous fish in spite of the fact that Runt transcription factors (Runx1–3 synonyms: Aml1– 3/Cbfa1–3/Pebp2aa–c) are central regulators of skeletal development in higher vertebrates [6,7]. They are characterized by a highly conserved DNA binding Runt domain and the presence of two promoters [8]. Each Runt gene has two isoforms with different N- termini starting with a MASNS-like motif under the distal P1 promoter and a MRIPV sequence under the proximal Promoter P2. Furthermore the 39 end has a conserved VWRPY-motif [8]. Runx2 is indispensable for osteogenesis as mice bearing a homozygous mutation in Runx2 completely lack bone [7], and Runx2 is together with Runx3 essential for cartilage differentiation [9,10]. Futhermore Runx2 directly regulates the key signaling molecule Indian hedehog (Ihh), which coordinates cartilage differentiation, endochondral ossification and limb outgrowth [10]. From the three members belonging to the mammalian Hedgehog (Hh) family (Ihh, Sonic hedgehog, PLoS Genetics | www.plosgenetics.org 1 March 2008 | Volume 4 | Issue 3 | e1000025