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First published online 16 May 2007
doi: 10.1242/dev.002618

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On the carapacial ridge in turtle embryos: its developmental origin, function and the chelonian body plan

¹ Laboratory for Evolutionary Morphology, Center for Developmental Biology (CDB), RIKEN, 2-2-3 Minatojima-minami, Kobe 650-0047, Japan.
² Sado Marine Biological Station, Faculty of Science, Niigata University, 87 Tassha, Sado, Niigata 952-2135, Japan.

View larger version (145K): [in this window] [in a new window]   Fig. 1. Development of the carapacial ridge in turtle embryos. (A,B) Comparison of the external morphology of TK stage 14 P. sinensis (A) and HH stage 26 chicken (B) embryos corresponding to the late pharyngula stage. Enlargement of the flank is shown on the right. In both of the embryos, longitudinal ridges appear on the lateral aspect of the flank (arrowheads). The ridge in P. sinensis represents the CR. (C) Comparison of transverse sections of a chicken-quail chimera (left), in which the somite has been replaced with that of a donor quail, and the HE-stained P. sinensis embryo (right), at comparable stages [for comparison of the developmental stages, see Nagashima (Nagashima et al., 2005)]. The longitudinal ridge in the chicken represents the Wolffian ridge caused by the folding of the proximal portion of the lateral body wall, or the somatopleure, which is also seen in the turtle. Arrows indicate the junction of the lateral body wall and the axial part of the embryonic body. Note that this junction corresponds to the boundary between the somite-derived and lateral-plate-derived dermis in the chimera. (D) Comparison of the developmental sequences of chicken (left column) and P. sinensis (right column) embryos. Transverse sections of the flank are shown for both animals. In the chicken, the indentation at the axial-lateral body wall junction (arrows) flattens out as development proceeds, whereas in P. sinensis, the junction remains visible because of the development of the CR, which arises just dorsal to the junction. Thus, the CR represents a ventrolateral limit of axial structure probably containing somite-derived mesenchyme. Scale bars: 1 mm for A,B; 100 µm for C,D. cr, carapacial ridge; hm, hypaxial muscles; r, rib; WR, Wolffian ridge.

View larger version (88K): [in this window] [in a new window]   Fig. 2. DiI labeling of the P. sinensis mesoderm and origin of the CR. (A-C) DiI was injected focally into the ventrolateral part of the dermomyotome at stages 11-11+. Arrowheads indicate the ventral limit of the CR or the junction of the lateral body wall. (A) Just after labeling. Note that the DiI labeling is restricted to the dermomyotome, and no part of the lateral plate is labeled. (B) Transverse sections of another embryo treated as in A and incubated for 5 days. Some somite-derived cells are migrating into the body wall, representing the hypaxial muscle precursor. (C) Higher magnification of the box in B. Mesenchymal labeling is restricted to the CR, except for a few labeled cells in the lateral body wall. (D-G) Labeling of the lateral plate (somatic) mesoderm. (D) A transverse section of the embryo just after labeling. Labeling is restricted to the most medial part of the lateral plate mesoderm, and no labeling occurs in the dermomyotome. (E-G) Five days after labeling. Transverse sections of an embryo treated as in D. Labeled mesenchyme is found only in the dermis of the lateral body wall. (F,G) Higher magnifications of boxes in E and F, respectively, showing the absence of labeled mesenchyme in the CR. In many embryos with the same labeling, a part of the CR-covering epidermis is labeled. (G) The CR does not contain any labeled mesenchymal cells. Scale bars: 100 µm for A,D; 200 µm for B,E; 50 µm for C,F,G. cr, carapacial ridge; dm, dermomyotome; hd, lateral body wall; hm, hypaxial muscle precursor; n, notochord; nt, neural tube; r, rib primordium; sc, sclerotome; sm, somatic mesoderm.

View larger version (46K): [in this window] [in a new window]   Fig. 3. Developmental patterning of the CR. (A) Schematic representation of the development at the trunk level in both species. Chicken and P. sinensis embryos show similar developmental patterns up to common stage 6 (Nagashima et al., 2005), when the proximal part of the lateral body wall swells laterally over the surface of the embryo at its junction with the axial part of the body (arrows), forming the Wolffian ridge. This junction is seen as an indentation on the lateral surface of the embryo, as is seen commonly in the amniote pharyngula. In the chicken embryo from HH stages 22 to 25, sclerotome-derived rib primordia and muscle plate invade the body wall, first as abaxial elements, and from stage 26 onwards, dorsal ribs and related muscles invade the body wall as primaxial elements. However, in P. sinensis, only the poorly developed hypaxial muscle is recognized as an abaxial element in the body wall, and axially developed ribs and muscles, although primaxial elements, never invade the body wall. The ventrolateral part of the axial domain also swells to form the CR adjacent to the Wolffian ridge dorsally. In the chicken, the lateral surface of the embryo flattens in later development. (B) Schematic representation of the axial domain and the lateral body wall in amniote embryos. (C). Differences in the expansion of the primaxial elements between turtles and other amniotes. Note that the muscle tissues are omitted from this scheme, and the lateral somitic frontier is shown by dotted lines. The primaxial ribs in amniotes correspond to the dorsal ribs of vertebrates; these arise initially in the axial domain of the embryonic body and grow secondarily ventrally into the body wall. In the turtle, the carapace is made of ribs that fail to invade the body wall. Thus, the growth pattern of the ribs and the primaxial dermis are co-extensive in the turtle. c=dr, carapace=dorsal ribs; da, dorsal aorta; dr, dorsal rib; hm, hypaxial muscle; lsf, lateral somitic frontier; mp, muscle plate; n, notochord; na, neural arch; nt, neural tube; sc, sclerotome; str, sternal rib; WR, Wolffian ridge.

View larger version (28K): [in this window] [in a new window]   Fig. 4. LEF-1 activity is essential to maintain the CR in P. sinensis. (A) Transverse section of the CR after electroporation of the GFP construct. Its expression is restricted to the epidermis. (B) Whole-mount embryo 4 days after the electroporation of GFP. No morphological changes are observed at the site of the operation (arrowhead). (C) Four days after the electroporation of dominant-negative LEF-1, a notch in the CR (arrowhead) appears at the site of electroporation, showing the local arrest of CR growth. Scale bars: 50 µm for A, 1 mm for B,C.

View larger version (72K): [in this window] [in a new window]   Fig. 5. Cauterization of the CR does not change the dorsoventral level of rib growth. (A-C) Transverse sections of a stage 14 P. sinensis embryo fixed just after unilateral microcauterization. (A) Enlargement of the experimental side of the embryo (B) shows necrosis of the CR tissue (arrowhead). (C) Enlargement of the left side of the same embryo showing the intact CR as the negative control. (D-G) In situ hybridization of an embryo 1 day after cauterization with a probe for CRABP1 (D,E) or for APCDD1 (F,G). The left panels represent the operated sides, and the right panels represent the corresponding control sides. In both cases, CR-specific gene expression is lost from the epidermis of the cauterized CR. (H-J) Three days after the microcauterization of the CR performed at stage 14. (H) A magnified view of the experimental side of the embryo; the image on the right shows an enlargement of the intact CR. Note that the intact CR comprises a thickened epidermis covering the accumulated mesenchyme (J), whereas on the operated side, these characteristic features of the CR have been lost (H). (I) Also note that the transversely viewed patterns of the ribs (r) are identical on the control and operated sides. (K-N) A whole-mount embryo 12 days after CR cauterization. Control (K) and operated (L) sides. Note the arrested growth of the carapacial margin at the site of cauterization (arrowhead). Dorsal (M) and lateral (N) views of the skeleton of the same embryo stained with Alcian Blue. Two successive ribs approximate each other distally (red arrowheads) at the site of CR cauterization (black brackets). Compare with the normal fan-shaped pattern on the control side (white brackets). Note that the ribs at the CR ablation do not grow ventrally across the level of the ablated CR (black arrowhead in N). (O-R) A whole-mount embryo 10 days after the cauterization of the apical ectodermal ridge of the forelimb. Control (O,P) and operated (Q,R) sides. (P,R) Alcian Blue-stained embryo. Note the severely disfigured autopod on the operated side (Q,R). Scale bars: 50 µm for A,C,H,J; 100 µm for B,D-G; 500 µm for I; 1 mm for K-R.

View larger version (90K): [in this window] [in a new window]   Fig. 6. CR does not induce the dorsal position but induces the fan-shape arrangement of the ribs. (A-M) An ectopic CR (cr), obtained from a stage 14- embryo, was labeled with DiI and grafted onto the host embryo dorsal to the endogenous CR (cr) at stage 13+. Observations were made of embryos 6 hours (stage 13+; A-D), 3 days (stage 15+; E-H) and 5 days (stage 17; I-M) after grafting. After 3 days of incubation, the grafted tissue integrated normally with the host tissue, and the expression of the CR-specific genes CRABP1 (G) and APCDD1 (H) was maintained in the graft. (J-M) Transverse histological sections at two different levels of the flank of the same embryo, as shown in I, stained with HE and Alcian Blue to visualize the cartilage. L and M are higher magnifications of the boxes in K. The ectopic CR is integrated completely into the host tissue and is associated with a thickened epidermis and underlying dense mesenchyme (L); the extracellular matrix is stained positively with Alcian Blue as the endogenous CR (M). Note in J and K that the pattern of rib growth has not changed on the experimental side and does not extend into the ectopic CR. (N) BrdU incorporation by the CR mesenchyme. BrdU was applied to a TK stage 16 embryo for 2 hours. Note the accumulation of BrdU at higher levels in the CR mesenchyme. (O) A schematic diagram showing the function of the CR in turtle embryos suggested by this study. In most amniote embryos (left, green), the ribs grow approximately parallel to each other compared with those in turtles (middle, red), which grow in a fan-like pattern resulting from the peripheral concentric growth of the carapacial primordium at the CR. Removing the CR arrests the growth of the carapacial primordium locally (right), and the anteroposterior growth of the vertebral column (purple arrow) causes the proximal parts of the ribs at the CR-ablated level (green) to become separated relative to the distal parts, resulting in the local disturbance of the distal parts of the ribs, as seen in Fig. 5M,N. Scale bars: 1 mm for A,E,I; 100 µm for B-D,F-H,N; 500 µm for J,K; 50 µm for L,M. fl, fore limb; hl, hind limb; m, myotome; n, notochord; nt, neural tube.

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