Comparative study of the shell development of hard- and soft-shelled turtles

Abstract

The turtle shell provides a fascinating model for the investigation of the evolutionary modifications of developmental mechanisms. Different conclusions have been put forth for its development, and it is suggested that one of the causes of the disagreement could be the differences in the species of the turtles used - the differences between hard-shelled turtles and soft-shelled turtles. To elucidate the cause of the difference, we compared the turtle shell development in the two groups of turtle. In the dorsal shell development, these two turtle groups shared the gene expression profile that is required for formation, and shared similar spatial organization of the anatomical elements during development. Thus, both turtles formed the dorsal shell through a folding of the lateral body wall, and the Wnt signaling pathway appears to have been involved in the development. The ventral portion of the shell, on the other hand, contains massive dermal bones. Although expression of HNK-1 epitope has suggested that the trunk neural crest contributed to the dermal bones in the hard-shelled turtles, it was not expressed in the initial anlage of the skeletons in either of the types of turtle. Hence, no evidence was found that would support a neural crest origin.

Keywords: carapace; marker genes; neural crest; plastron; turtles.

Fig. 1

The turtle shell. Inside views of the carapacial (left) and plastral (right) skeletons of a hard-shelled turtle, Trachemys scripta elegans (carapace length 7.3 cm). ent, entoplastron; epi, epiplastron; hyo, hyoplastron; hypo, hypoplastron; nu, nuchal plate; pe, peripheral plate; py, pygal plate; r, dorsal ribs; spy, suprapygal plate; v, dorsal vertebrae; xip, xiphiplastron.

Fig. 2

Development of Trachemys scripta. (Left) Left lateral view of the embryos. (Middle) Forelimb. (Right) Hindlimb. In G stage 14, carapacial ridge (CR) appears as a longitudinal ridge in the flank (arrowheads), and in stage 15, the CR acquires a segmental pattern (arrowheads). The ridge comprises the margin of the carapace in the latter stages. The morphology of the forelimb bud is comparable to that in Chelydra serpentina (Yntema, 1968) and Pelodiscus sinensis (Tokita & Kuratani, 2001) at the same stage. Namely, in G stage 13, the length is longer than the width, and G stage 14 is an early paddle stage with a digital plate, in G stage 15 the digital plate is well formed without a digital groove, in G stage 16 digital ridges appear, in G stage 17 the periphery of the digital plate shows a slight serration and five digits are apparent, and in G stage 18 the periphery of the digital plate is serrated deeply, similar to that of maple leaves. Scale bars: 1 mm (left column), 200 μm (middle and right columns).

Fig. 3

Expressions of genes related to the carapace development in Trachemys scripta. (A–F) Expressions of Fgfs. (A,B) Positive controls. In G stage 13 embryo, Fgf10 (A) and Fgf8 (B) are expressed in the otic vesicle (ov) and apical ectodermal ridge (aer) in the hindlimb bud, respectively. (C–F) Fgf10 (C,D) and Fgf8 (E, F) expressions in the carapacial ridge (cr) and its adjacent area. (E, F) Adjacent sections to (C,D), respectively. Note that both in G stage 14 (C,E) and 15 (D,F), these genes are not expressed in the CR and ribs, whereas AER is positive for the Fgf8 probe (E). (G–I) Expressions of Wnt-related genes. (H) Adjacent section to (G). Wnt5a (G) and APCDD1(H) are expressed in the mesenchyme of the CR, whereas HGF (I) is expressed in the lateral sclerotome (sc). afg, acoustico-facialis ganglion; c, coelom; mp, muscle plate; n, notochord. Scale bars: 100 μm (A,C–F), 50 μm (B, G–I).

Fig. 4

Three-dimensional reconstruction of the shoulder region of G stage 17 Trachemys scripta embryo. (Left) Lateral view. Rostral is on the right. Note that the shoulder girdle is rostro-lateral to the muscle plate. (Right) Caudal view. Medial is on the left. The left half of the embryo is eliminated. Note that ribs grow along the muscle plate, which is folded medially around the shoulder girdle. ac, acromion; as, serratus anterior; cor, coracoid; g, glenoid cavity; pl, plastron; sc, scapular blade.

Fig. 5

Expression of the HNK-1 epitope in Trachemys scripta embryos. Transverse sections of T. scripta embryos are either stained with HNK-1 and counterstained with hematoxylin (A–G,I,K) or simply stained with hematoxylin and eosin (HE) and then Alcian blue (H,J,L). (A–C) Transverse sections at low magnification. (D–L) Higher magnification of the boxes in (A–C). (H,J, L) Adjacent sections to (G,I,K), respectively. (D–F) The dorsal part of the embryo showing the distribution of the HNK-1 epitope. Note that the HNK-1 epitope appears from G stage 17 (E) and the expression domain expands in G stage 18 (F). (G–J) Distribution of the HNK-1 epitope in the ribs. A part of the mesenchyme surrounding the ribs is HNK-1-positive (G,H). In G stage 18 (I,J), the epitope was observed in the cells with a round nucleus in the periosteum (arrowheads), whereas the cells with a long nucleus are HNK-1-negative (arrows). (K,L) Distribution of the HNK-1 epitope in the plastron of the G stage 18 embryo. Immunoreactivity was observed for the osteoblast in the plastral bones and in the adjacent mesenchyme. drg, dorsal root ganglion; fl, fore limb; na, neural arch; nt, neural tube. Scale bars: 500 μm (A–C), 100 μm (D–F), 50 μm (G,H,K,L), 20 μm (I,J).

Fig. 6

Expression of the HNK-1 epitope in Trachemys scripta embryos. Transverse sections of T. scripta embryos were either stained with HNK-1 and counterstained with hematoxylin (A,C) or simply stained with HE and then Alcian blue (B,D). (B,D) are adjacent sections to (A,C), respectively. (A, B) Higher magnification of the box (asterisk) in Fig. 5B, showing distribution of the HNK-1 epitope in the initial anlage of the plastral bones in the G stage 17 embryo. Note that the anlage is HNK-1-negative, whereas the peripheral nerves are strongly stained with the antibody (arrowheads). (C,D) Distribution of the HNK-1 epitope in the humerus (h) of the G stage 18 embryo. Note that the expression domain of HNK-1 overlaps the area of the intramembranous bone (arrows). Scale bars: 50 μm.

Fig. 7

Expression of the HNK-1 epitope in Pelodiscus sinensis and Gallus gallus embryos. Transverse sections of P. sinensis (A–I) and G. gallus (J,K) embryos were either stained with HNK-1 and counterstained with hematoxylin (A,C,D,F,H,J) or simply stained with HE (B) and then Alcian blue (E,G,I,K). (B,E,G,I, K) Adjacent sections to (A,D,F,H,J), respectively. (A–I) Transverse sections of P. sinensis. (A,B) TK stage 11 embryo. Cell population dorso-lateral to the neural tube is stained with the antibody. From its position, these cells appear to be neural crest cells. (C) The dorsal part of the TK stage 18 embryo, showing distribution of the HNK-1 epitope. Note that the dorsal mesenchyme is negative for the antibody, which recognizes only the spinal cord (nt), peripheral nerves (arrowheads) and intrinsic back muscles (bm). (D–I) Distribution of the HNK-1 epitope in the intramembranous bones. (D,E) and (F–I) are TK stage 18 and 20, respectively. Note that the intramembranous bones (arrows) in the plastral bone (D,E), ribs (F,G) and humerus (H,I) of P. sinensis are negative for the antibody. (J and K) Distribution of the HNK-1 epitope in the humerus of HH 37 G. gallus embryo. The intramembranous bone (arrows) is negative for the antibody. Arrowheads show peripheral nerves for the positive control. a, dorsal aorta; d, dermomyotome; sc, sclerotome. Scale bars: 50 μm.