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).
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