Development of the carapacial ridge: implications for the evolution of genetic networks in turtle shell development Jacqueline E. Moustakas Department of Integrative Biology and Museum of Paleontology, University of California, 1101 Valley Life Sciences Building, Berkeley, CA 94720, USA Correspondence (email: moustakas@berkeley.edu) SUMMARY Paleontologists and neontologists have long looked to development to understand the homologies of the dermal bones that form the ‘‘armor’’ of turtles, crocodiles, armadillos, and other vertebrates. This study shows molecular evidence supporting a dermomyotomal identity for the mesenchyme of the turtle carapacial ridge. The mesenchyme of the carapace primordium expresses Pax3, Twist1, Dermo1, En1, Sim1, and Gremlin at early stages and before overt ossification expresses Pax1. A hypothesis is proposed that this mesenchyme forms dermal bone in the turtle carapace. A comparison of regulatory gene expression in the primordia of the turtle carapace, the vertebrate limb, and the vertebral column implies the exaptation of key genetic networks in the development of the turtle shell. This work establishes a new role for this mesodermal compartment and highlights the importance of changes in genetic regulation in the evolution of morphology. INTRODUCTION Dermal bone is an important component of the vertebrate skeleton that makes up the majority of the skull, and in some lineages also forms a postcranial bony armor. The bone- forming osteoblasts of dermal bone differentiate directly from mesenchymal precursor cells in the dermis of the skin, unlike the axial skeleton, which develops through a cartilage model. The mesenchyme that forms the dermal bones of the cranium of extant vertebrates is derived from the neural crest (Couly et al. 1993; Jiang et al. 2002; Gross and Hanken 2005), but the developmental origin of the mesenchyme of postcranial der- mal bone has remained elusive. One well-known, but poorly understood example of post- cranial dermal bone in vertebrates is the turtle shell. The turtle shell appeared over 200 Ma (Gaffney et al. 1987), but uncer- tainty about the origins of turtles (Lee 1997; Rieppel and Reisz 1999) has prevented a clear understanding of its origin and evolution. The turtle shell is composed of a dorsal armor (carapace) and a ventral armor (plastron). Previous work has shown that, as in the skull, the bones of the plastron are derived from the neural crest (Cebra-Thomas et al. 2007). Here, developmental genetic data are used to compare the development of the embryonic turtle carapace with other ver- tebrate skeletal systems. The first morphological indication of carapace develop- ment is seen at stage Yntema 14 (Y14; Yntema 1968) with the appearance of the carapacial ridge (CR) along the lateral trunk of the turtle embryo (Ruckes 1929; Burke 1989; Fig. 1). The CR is a protrusion of mesenchyme cells encased in an overlying thickened surface ectoderm that will form the lateral margin of the turtle carapace. This structure appears similar morphologically to the primordium of the vertebrate limb (Burke 1989). Extirpation and labeling experiments have shown that somitic mesoderm contributes mesenchyme to the CR (Yntema 1970; Nagashima et al. 2007), whereas the me- senchyme of the limb bud is derived from the lateral plate mesoderm (reviewed in Martin 1998). Gene expression was studied during the morphogenesis of the CR in the red-eared slider turtle Trachemys scripta elegans in the somite compartments that could contribute me- senchyme to the carapace. During embryogenesis, epithelial somites form as segmental units of the paraxial mesoderm on either side of the neural tube and notochord. The ventral half of the epithelial somite undergoes an epithelial to me- senchymal transition to form the sclerotome. The sclerotome gives rise to the vertebrae, ribs, and meninges of the spinal cord (Christ et al. 2004). The dorsal half of the somite remains epithelial during the de-epithelialization of the sclerotome and is known as the dermomyotome. The dermomyotome con- tains the precursors for the dermis, epaxial, and hypaxial muscle, as well as the bone of the scapular blade (Scaal and Christ 2004). Studies in several model systems have shown that the paired-box (Pax) transcription factor Pax3 is EVOLUTION & DEVELOPMENT 10:1, 29–36 (2008) & 2008 The Author(s) Journal compilation & 2008 Blackwell Publishing Ltd. 29