The Carapacial Ridge and the Entry of the Ribs into the Dermis

The formation of the carapace involves several steps. The first concerns the entry of the rib precursor cells into the dermis. The turtle egg is laid at the mid-gastrula stage. Turtle gastrulation has not been studied in detail for almost eight decades and presents an interesting contrast to the wellstudied avian system (see review; Gilland & Burke, 2004). Later stages of nerulation and somite formation are similar to those processes in the chick (Ewert, 1985; Pasteels, 1937, 1957). The first sign that the organism is to become a turtle rather than some other tetrapod occurs at Yntema stage 14/Greenbaum stage 15 (Yntema, 1968—stages are for Chelydra; Greenbaum, 2002—stages are for Trachemys. Stage 14/15 is approximately equivalent to Hamburger–Hamilton chick stage 24).

At this stage are the first signs of ridges on the lateral surfaces of the embryo, dorsal to the limb buds (Ruckes, 1929). At first, these ridges are seen between the two limb buds, and only later do the ridges extend anteriorly and posteriorly. This structure has been named the carapacial ridge (CR) (Burke, 1989b, 1989c, 1991), and the paired carapacial ridges will eventually form the outer edge of the carapace. The CR is formed by a thickening of the ectoderm and is underlaid by a condensed somite-derived mesenchyme (Yntema, 1970; Burke, 1989b, 1989c; Nagashima et al., 2005). Ruckes’ (1929) observations of turtle embryos described two important features of turtle shell development. First, there is an accelerated lateral growth of the dorsal dermis of the trunk compared to growth in the dorso-ventral plane. Second, there is an apparent ‘‘ensnarement’’ of the growing ribs by the dermis. The involvement of the ribs with the carapacial dermis results in their growth in a predominantly lateral direction (Figure 1.1A). The limb girdles develop in typical tetrapod fash- ion but because of the growth trajectory of the ribs, the pectoral girdle becomes ventral and deep to the axial elements. Yntema (1970) performed a series of somite extirpation experiments on snapping turtles, confirming a somitic origin for the ribs and dermis of the carapace. Post-otic somite pairs 12 through 21 are involved in forming the carapace in Chelydra.

In 1989, Burke proposed that the thickened ectoderm and condensed mesenchyme of the CR is typical of sites of epithelial-mesenchymal interactions. The distributions of the cell adhesion proteins fibronectin and N-CAM in the CR are similar to their locations in other inductive sites such as the early limb bud or feather primordia. Burke (1991) tested the causal relationship between the CR and the growth trajectory of the ribs. In the first set of experiments, she removed the CR by tungsten needles from one side of stage 1 through stage 16 embryos. These extirpations included both ectodermal and mesenchymal components. In those cases where the CR did not regenerate, the growth trajectory of the rib was deflected toward a neighboring region that did have a CR. In a second set of experiments, she placed tantalum barriers between the somite and the presumptive CR.

The surviving embryos showed disruptions such that where the CR was interrupted, entire regions of the dermal carapace were missing. The ribs associated with these missing regions interdigitated with those bones of the plastron. Burke concluded that the normal development of the ribs appears to be directed by the CR. In the absence of the CR, these ribs project ventrally into the lateral plate mesoderm like the ribs of non-Chelonian vertebrates.

Loredo and colleagues (2001) were the first to analyze the CR with molecular probes and found fibroblast growth factor-10 (FGF-10) expression in the mesenchyme condensed beneath the Trachemys CR. Fibroblast growth factors are paracrine factors that are critical in the patterning, migration, and differentiation of numerous cell types, and they are especially important in determining the fates of cells in the face and in the limbs. Vincent and coworkers (2003) found the turtle homologue of transcription factor msx1 is expressed in the mesenchyme of the Emys CR. This result furthered the notion that the CR was made through mesenchymal/epithelial interactions similar to those that generate the limb bud. The Wnt signaling pathway is used in several embryonic inductions and can mediate the effects of fibroblast growth factors (in the limb bud). By using RT-PCR, Kuraku and colleagues (2005) found turtle orthologs of Sp5 and Wnt targets APDCC-1 and LEF-1 in the CR mesenchyme and ectoderm of the Chinese softshell turtle Pelodiscus. They also found CRABP-1 expressed in the CR ectoderm. However, they did not detect the expression of either of the previously reported genes, msx1, or FGF-10 in the CR mesenchyme of this species. Species differences might be important in these patterns because the costal bones of Pelodiscus might form by different methods from that of the hardshell turtles (Zangerl, 1969), and the pattern of FGF-10 distribution in the limbs of Pelodiscus differed from the expression pattern seen in the limbs of Trachemys.

The FGF family of paracrine factors is often involved in chemotaxis, and in the chick limb, FGF-10 appears to be critical in directing the endodermal chemotaxis in the lung (Park et al., 1998; Weaver et al., 2000). Cebra-Thomas and colleagues (2005) demonstrated that FGF-induced chemotaxis plays an important role in causing the rib precursors to enter the CR. They cultured eviscerated trunk explants of stage 15 Trachemys embryos ventral-side down on nucleopore membranes.

At this stage, the CR is visible and the sclerotome has been specified. After three days in culture, the ribs have migrated into the CR, and the ridges are visibly raised. However, if SU5402 (an inhibitor of FGF signaling) is added to the culture media when the explants are established, the CR degenerates and the ribs travel ventrally, like the ribs of non-Chelonians. Cebra-Thomas and colleagues also show that chick rib precursor cells are responsive to FGF-10, and beads containing FGF-10 will redirect chick rib growth in culture. Thus, the CR appears to be critical for directing the migration of rib precursor cells into it. FGF signaling in the CR appears to be crucial in the maintenance of the CR and is either directly or indirectly responsible for guiding the rib precursor cells into the CR.

Another finding of Cebra-Thomas and colleagues (2005) was that the distal tip of each rib expressed FGF-8. High levels of FGF-8 expression have not been reported in the distal ribs of other organisms. Cebra-Thomas and colleagues speculate that FGF-8 (in the ribs) and FGF-10 (in the CR mesenchyme) may establish a positive feedback loop such that the growth of the rib becomes coordinated with the growth of the carapace. Such a positive feedback loop has been shown to be responsible for the coordinated outgrowth of the chick and mouse limb buds (Ohuchi et al., 1997; Kawakami et al., 2001).

How the Turtle Gets Its Shell

FIGURE  .   Development of the carapace. (A). Entry of cartilaginous rib precursor (arrow) into carapa-

cial ridge of Trachemys embryo around stage 16. The following show bone formation in Trachemys scripta, 

stained with Alcian blue (cartilage) and alizarin red (bone). (B) 1.2-cm embryo showing cartilaginous ribs 

forming the outline of the shell. (C). Ventral view of 3.1-cm carapace, showing intramembranous ossification 

of the nuchal bone and around and in the anterior ribs. (D) Lateral view of the same carapace, showing region 

of rib chondrogenic growth (blue, arrow) and transition zone (white) between cartilage and bone (red). (E) 

Dorsal view of 118-day (CL = 3.1 cm) hatchling carapace showing expanded nuchal bone region, the fusion 

of the anterior costal ossification centers, and the peripheral bone ossification centers that start anteriorly. 

The pigmentation of the epidermal scutes can be seen. (F) Dorsal view of 185-day (CL = 4.5 cm) hatchling 

carapace showing fusion of marginal ossification regions anteriorly, as well as the pygal ossification center 

posteriorly. The costal ossification centers have created bony armor dorsally (the blue staining is beneath the 

carapace). (G) Predominant pattern of the adult carapacial bones. (Modified from Gilbert et al., 2001; G modi-

fied from Zangerl, 1969.)

The unpaired midline dermal bones of the carapace, called neurals, are fused with the neural spines of the 10 thoracic vertebrae (Figure 1.1). The costal bones extend from the neurals toward the periphery. There are eight pairs and each is intimately associated with a rib (Figure 1.1E). Gennrally, there is a one-to-one correspondence between the vertebral spines and the neural bones, and How the Turtle Gets Its Shell

between the ribs and the costal bones of the carapace. This relationship does not hold in the anterior and posterior ends of the shell, where the vertebral centra are shortened and have little or no contact with the shell. The first costal bone overlies ribs one and two, and the eighth overlies ribs nine and ten (variants have nine pairs of costal bones). The pygal and suprapygal bones form the rear of the carapace. These bones have no contact with vertebra and ribs but project over the sacrum and pelvis. The peripheral bones form the edge of the carapace. There are generally 11 pairs of peripheral bones; before making contact with the costals, they form a socket around the distal tip of ribs two through nine. The nuchal bone forms the anterior margin of the carapace, which overhangs but is not attached to the posterior cervical vertebra. This bone extends laterally around the margins of the carapace to the level of the second rib. It is overlaid by the first three peripheral bones laterally and contacts the first costals and neural bone posteriorly. Each of the carapacial bones is connected by sutures to its neighbors. The distal edge of each costal is attached by suture to the peripheral bones. This contact often does not occur until later stages of post-hatching growth, leaving open a peripheral ring of fontanels that surround the distal tips of the ribs.

Sections across the carapaces of adult turtles show a three-layered arrangement of the bone. The central portion of the bone is a spongy layer containing spherical cavities. On either side of the spongy layer are layers of more compact lamellar bone. This compact bone is thought to form beneath the inner and outer periosteal membranes. The shapes and relative sizes of these bony regions determine the general form of the shell in different genera (Yntema, 1970; Ewert, 1985; Cherepanov, 1997).

ANATOMY OF THE TURTLE SHELL

ANATOMY OF THE TURTLE SHELL

The character and homology of the bony elements of the turtle shell have a long history of controversy. The shell is comprised of the endochondral axial elements of the trunk overlaid by a mosaic of dermal bones and an outer epidermal layer made of keratinous scales (also called scutes or shields). All turtles possess 10 trunk vertebrae associated with the carapace. Each vertebra possesses a single-headed rib that often shares an articulation with the next anterior vertebra. The first and tenth ribs are diminutive and normally extend a short distance before making contact with the second and ninth ribs, respectively. The tenth rib is often indistinguishable in both embryos and adults, but the presence of a large tenth rib in embryos is a normal variation. The thoracic ribs enter the dermis of the shell a short distance from their articulation with the vertebrae, and they extend laterally within the carapacial dermis, terminating at the periphery (reviewed by Zangerl, 1969). In the dermal layer of the shell, there are generally 59 bones: the carapace has 38 paired and 12 or 13 unpaired bones (sometimes the suprepygeal bone is divided and sometimes it is not). The plastron contains one unpaired and eight paired bones. With the exception of a few key taxa, the only real variations in this general scheme occur as individual variations around the neck and tail where the axial skeleton is not closely joined to the carapace. The shapes and relative sizes of the bones determine the general form of the shell in different genera.

The shell’s epidermal layer generally consists of 38 scutes in the carapace and 16 in the plastron. However, this can vary depending on the shape of the shell (domed, hinged, flapped, and so on; see Chapter 3). The shield and bone patterns are not in register; each shield covers a particular area of the bony mosaic. The pattern of the sulci that form between neighboring scutes and the sutures that form between neighboring bones form two minimally overlapping patterns. The epidermal shield pattern develops long before the shell bones begin to ossify, and the underlying dermis may play a major role in the formation of the epidermal scutes, similar to the influence of somitic dermis of feather patterns in the chick (Yntema, 1970; Cherepanov, 1989; Alibardi & Thompson, 1999a,b).

The turtle shell

The turtle shell is a remarkable evolutionary novelty that defines the order Chelonia. The turtle shell is found in three general forms based on the nature and degree of ossification: hardshells, softshells, and leatherbacks. This section will concentrate almost totally on the bony component of those shells of the hardback turtles of the Emys and Chelydae families. This shell is composed of two main parts, the dorsal carapace and the ventral plastron, connected along the midflanks by lateral bridges.
Altogether, the shell contains over 50 dermal bones that are homologous to no other bone in anyother vertebrate order. Moreover, the presence of this bony casing has necessitated extensive modifications of the tetrapod body plan (Zangerl, 1969). Whereas dermal ossification itself is a primitivecharacter for vertebrates (Smith & Hall, 1993), the turtle shell represents an extreme developmentof the dermal skeleton among tetrapods.

The shell clearly has adaptive value for turtles as physical protection, but it also serves physiological functions in different species as a site of hematopoiesis, a reservoir for water, fat, or wastes,and a buffer for pH. The embryonic development of the shell involves a dramatic hypertrophy of thedermis in the dorsal body wall and a resultant rearrangement of the typical relationship between thepectoral girdle and the axial skeleton. Thus, turtles are the only vertebrates whose limbs are founddeep to the ribs.

The paraxial and limb-girdle musculature—the neck and skull—are also greatlymodified. As we detail here, the key innovation for the chelonians appears to be the carapacialridge, a bulge of ectoderm and mesoderm that influences the growth of the ribs (Burke, 1989a). Theribs are enveloped within the dorsal dermis, resulting in their lateral displacement as the dermis rapidly expands. Thus instead of extending ventrally and enclosing the thoracic cavity, the turtle ribs become integrated into the carapacial dermis.

The neural arches of the vertebrae also fuse with the midline of the carapace. As the anonymous author (1676) of the letter to the Royal Society of London wrote in 1676:“The Anatomie of a Tortoise, showing that what were the Ribs in other Animals one upper Shell is in the Tortoise, and that to that upper Shell are firmly fastened the spinal Vertebrae, so that the Animal cannot go out of its Home, as Snails do.”