Evolution of a Core Gene Network for Skeletogenesis in Chordates

Jochen Hecht,Sigmar Stricker,Siegfried Ehrich,Stefan Mundlos,Albert J Poustka,Lars Podsiadlowski,Ulrike Wiecha,Volkhard Seitz,Christoph Dieterich,Asita Stiege,Julia Suvorova,Georgia Panopoulou,Sebastian Shimeld

doi:10.1371/journal.pgen.1000025

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.

Highlights

The skeleton is a hallmark of vertebrates and has been widely used over the past decades for phylogenetic analyses [1]
In this study we show that the stem species of chordates harboured a single Runt gene, whereas three Runt genes were present before the emergence of gnathostomes
Using quantitative Reverse Transcriptase PCR from dissected tissues we found that the MgRunxA gene had its highest expression in hard cartilage, followed by the gill region and soft cartilage (Figure 3)

Summary

Introduction

The skeleton is a hallmark of vertebrates and has been widely used over the past decades for phylogenetic analyses [1]. 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 the characteristic matrix protein for jawed vertebrates (gnathostomes) [3,4]. 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]

Methods

Results

Conclusion