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Molecular footprinting of skeletal tissues in the catshark Scyliorhinus canicula and the clawed frog Xenopus tropicalis identifies conserved and derived features of vertebrate calcification.

Enault S, Muñoz DN, Silva WT, Borday-Birraux V, Bonade M, Oulion S, Ventéo S, Marcellini S, Debiais-Thibaud M - Front Genet (2015)

Bottom Line: Understanding the evolutionary emergence and subsequent diversification of the vertebrate skeleton requires a comprehensive view of the diverse skeletal cell types found in distinct developmental contexts, tissues, and species.To date, our knowledge of the molecular nature of the shark calcified extracellular matrix, and its relationships with osteichthyan skeletal tissues, remain scarce.Finally, we uncover a striking parallel, from a molecular and histological perspective, between the vertebral cartilage calcification of both species and discuss the evolutionary origin of endochondral ossification.

View Article: PubMed Central - PubMed

Affiliation: Institut des Sciences de l'Evolution de Montpellier, UMR5554, Université Montpellier, Centre National de la Recherche Scientifique, IRD, EPHE Montpellier, France.

ABSTRACT
Understanding the evolutionary emergence and subsequent diversification of the vertebrate skeleton requires a comprehensive view of the diverse skeletal cell types found in distinct developmental contexts, tissues, and species. To date, our knowledge of the molecular nature of the shark calcified extracellular matrix, and its relationships with osteichthyan skeletal tissues, remain scarce. Here, based on specific combinations of expression patterns of the Col1a1, Col1a2, and Col2a1 fibrillar collagen genes, we compare the molecular footprint of endoskeletal elements from the chondrichthyan Scyliorhinus canicula and the tetrapod Xenopus tropicalis. We find that, depending on the anatomical location, Scyliorhinus skeletal calcification is associated to cell types expressing different subsets of fibrillar collagen genes, such as high levels of Col1a1 and Col1a2 in the neural arches, high levels of Col2a1 in the tesserae, or associated to a drastic Col2a1 downregulation in the centrum. We detect low Col2a1 levels in Xenopus osteoblasts, thereby revealing that the osteoblastic expression of this gene was significantly reduced in the tetrapod lineage. Finally, we uncover a striking parallel, from a molecular and histological perspective, between the vertebral cartilage calcification of both species and discuss the evolutionary origin of endochondral ossification.

No MeSH data available.


Related in: MedlinePlus

Cartilage calcification and collagen expression in Scyliorhinus canicula vertebrae. (A–D) Transverse sections of the vertebrae of 6 cm-long embryos (black arrowheads show the hyaline cartilage of the neural arches). (A) Alcian blue and Alizarin red double staining revealing the distribution of the hyaline cartilage and the absence of detectable calcification. (B–D)In situ hybridizations showing the expression of Sc-Col2a1, Sc-Col1a1, and Sc-Col1a2, as indicated. (E) Schematic drawing of the vertebral anatomy from 9 cm-long S.c. embryos (lateral view) and of the orientation of the transverse sections (blue dotted line) represented in (F) and shown in (I–O'). (G) General histology of the centrum. (H) General histology of the neural arches. (I) Alizarin red and Alcian blue double staining. (J,K) HES staining of the centrum and of the neural arch. (L–N)In situ hybridizations showing the expression of Sc-Col2a1, Sc-Col1a1, and Sc-Col1a2, as indicated. Arrowheads in (L',M') indicate scattered Sc-Col1a1 and Sc-Col1a2 positive cells embedded in the calcified layer of the neural arches. (O) Immunofluorescence using an anti-Type II collagen (Col2) specific antibody. Higher magnifications of (I,L–O) are shown in (I',L'–O') respectively. Orange and black arrowheads show the calcifying matrix of the centrum and neural arches, respectively. Cc, chordocytes; Ch, chondroctyces; na, neural arch; nac, neural arch cartilage; ns, notochord sheath; nt, neural tube; ntc, notochord core; Pe, perichondrium; vb, vertebral body; vbc, vertebral body cartilage. Insets in (L–O) are shown at higher magnification in (L'–O'), respectively. Scale bars: (A–D) 250 μm; (I,L–O) 200 μm; (J,K) 50 μm.
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Figure 2: Cartilage calcification and collagen expression in Scyliorhinus canicula vertebrae. (A–D) Transverse sections of the vertebrae of 6 cm-long embryos (black arrowheads show the hyaline cartilage of the neural arches). (A) Alcian blue and Alizarin red double staining revealing the distribution of the hyaline cartilage and the absence of detectable calcification. (B–D)In situ hybridizations showing the expression of Sc-Col2a1, Sc-Col1a1, and Sc-Col1a2, as indicated. (E) Schematic drawing of the vertebral anatomy from 9 cm-long S.c. embryos (lateral view) and of the orientation of the transverse sections (blue dotted line) represented in (F) and shown in (I–O'). (G) General histology of the centrum. (H) General histology of the neural arches. (I) Alizarin red and Alcian blue double staining. (J,K) HES staining of the centrum and of the neural arch. (L–N)In situ hybridizations showing the expression of Sc-Col2a1, Sc-Col1a1, and Sc-Col1a2, as indicated. Arrowheads in (L',M') indicate scattered Sc-Col1a1 and Sc-Col1a2 positive cells embedded in the calcified layer of the neural arches. (O) Immunofluorescence using an anti-Type II collagen (Col2) specific antibody. Higher magnifications of (I,L–O) are shown in (I',L'–O') respectively. Orange and black arrowheads show the calcifying matrix of the centrum and neural arches, respectively. Cc, chordocytes; Ch, chondroctyces; na, neural arch; nac, neural arch cartilage; ns, notochord sheath; nt, neural tube; ntc, notochord core; Pe, perichondrium; vb, vertebral body; vbc, vertebral body cartilage. Insets in (L–O) are shown at higher magnification in (L'–O'), respectively. Scale bars: (A–D) 250 μm; (I,L–O) 200 μm; (J,K) 50 μm.

Mentions: The transverse sections of 6 cm embryos shown in Figures 2A–D reveal that the S.c. vertebrae are cartilaginous, devoid of calcification, and express Sc-Col2a1 (in chondrocytes of the centrum and the neural arches) and Sc-Col1a1 and Sc-Col1a2 (in the perichondrium surrounding all vertebral elements). In the vertebral column of 7 cm-long embryos, Alcian blue stains the cartilaginous vertebrate body and the neural arches (Figures 2E–I'). Alizarin red specifically stains the fibrous perichondrium of the neural arches as well as an internal calcification ring located within the centrum and surrounding the notochord, as reported in other chondrichthyan species (see Figures 2E–K and Peignoux-Deville et al., 1982; Eames et al., 2007). Histologically, the calcified ring of the vertebral body exhibits darker HES staining of the matrix surrounding large cells of chondrocytic appearance (Figures 2G,J). By contrast, cells located in the calcifying extracellular matrix of the neural arches are thin with reduced amount of cytoplasm (Figures 2H,K).


Molecular footprinting of skeletal tissues in the catshark Scyliorhinus canicula and the clawed frog Xenopus tropicalis identifies conserved and derived features of vertebrate calcification.

Enault S, Muñoz DN, Silva WT, Borday-Birraux V, Bonade M, Oulion S, Ventéo S, Marcellini S, Debiais-Thibaud M - Front Genet (2015)

Cartilage calcification and collagen expression in Scyliorhinus canicula vertebrae. (A–D) Transverse sections of the vertebrae of 6 cm-long embryos (black arrowheads show the hyaline cartilage of the neural arches). (A) Alcian blue and Alizarin red double staining revealing the distribution of the hyaline cartilage and the absence of detectable calcification. (B–D)In situ hybridizations showing the expression of Sc-Col2a1, Sc-Col1a1, and Sc-Col1a2, as indicated. (E) Schematic drawing of the vertebral anatomy from 9 cm-long S.c. embryos (lateral view) and of the orientation of the transverse sections (blue dotted line) represented in (F) and shown in (I–O'). (G) General histology of the centrum. (H) General histology of the neural arches. (I) Alizarin red and Alcian blue double staining. (J,K) HES staining of the centrum and of the neural arch. (L–N)In situ hybridizations showing the expression of Sc-Col2a1, Sc-Col1a1, and Sc-Col1a2, as indicated. Arrowheads in (L',M') indicate scattered Sc-Col1a1 and Sc-Col1a2 positive cells embedded in the calcified layer of the neural arches. (O) Immunofluorescence using an anti-Type II collagen (Col2) specific antibody. Higher magnifications of (I,L–O) are shown in (I',L'–O') respectively. Orange and black arrowheads show the calcifying matrix of the centrum and neural arches, respectively. Cc, chordocytes; Ch, chondroctyces; na, neural arch; nac, neural arch cartilage; ns, notochord sheath; nt, neural tube; ntc, notochord core; Pe, perichondrium; vb, vertebral body; vbc, vertebral body cartilage. Insets in (L–O) are shown at higher magnification in (L'–O'), respectively. Scale bars: (A–D) 250 μm; (I,L–O) 200 μm; (J,K) 50 μm.
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Figure 2: Cartilage calcification and collagen expression in Scyliorhinus canicula vertebrae. (A–D) Transverse sections of the vertebrae of 6 cm-long embryos (black arrowheads show the hyaline cartilage of the neural arches). (A) Alcian blue and Alizarin red double staining revealing the distribution of the hyaline cartilage and the absence of detectable calcification. (B–D)In situ hybridizations showing the expression of Sc-Col2a1, Sc-Col1a1, and Sc-Col1a2, as indicated. (E) Schematic drawing of the vertebral anatomy from 9 cm-long S.c. embryos (lateral view) and of the orientation of the transverse sections (blue dotted line) represented in (F) and shown in (I–O'). (G) General histology of the centrum. (H) General histology of the neural arches. (I) Alizarin red and Alcian blue double staining. (J,K) HES staining of the centrum and of the neural arch. (L–N)In situ hybridizations showing the expression of Sc-Col2a1, Sc-Col1a1, and Sc-Col1a2, as indicated. Arrowheads in (L',M') indicate scattered Sc-Col1a1 and Sc-Col1a2 positive cells embedded in the calcified layer of the neural arches. (O) Immunofluorescence using an anti-Type II collagen (Col2) specific antibody. Higher magnifications of (I,L–O) are shown in (I',L'–O') respectively. Orange and black arrowheads show the calcifying matrix of the centrum and neural arches, respectively. Cc, chordocytes; Ch, chondroctyces; na, neural arch; nac, neural arch cartilage; ns, notochord sheath; nt, neural tube; ntc, notochord core; Pe, perichondrium; vb, vertebral body; vbc, vertebral body cartilage. Insets in (L–O) are shown at higher magnification in (L'–O'), respectively. Scale bars: (A–D) 250 μm; (I,L–O) 200 μm; (J,K) 50 μm.
Mentions: The transverse sections of 6 cm embryos shown in Figures 2A–D reveal that the S.c. vertebrae are cartilaginous, devoid of calcification, and express Sc-Col2a1 (in chondrocytes of the centrum and the neural arches) and Sc-Col1a1 and Sc-Col1a2 (in the perichondrium surrounding all vertebral elements). In the vertebral column of 7 cm-long embryos, Alcian blue stains the cartilaginous vertebrate body and the neural arches (Figures 2E–I'). Alizarin red specifically stains the fibrous perichondrium of the neural arches as well as an internal calcification ring located within the centrum and surrounding the notochord, as reported in other chondrichthyan species (see Figures 2E–K and Peignoux-Deville et al., 1982; Eames et al., 2007). Histologically, the calcified ring of the vertebral body exhibits darker HES staining of the matrix surrounding large cells of chondrocytic appearance (Figures 2G,J). By contrast, cells located in the calcifying extracellular matrix of the neural arches are thin with reduced amount of cytoplasm (Figures 2H,K).

Bottom Line: Understanding the evolutionary emergence and subsequent diversification of the vertebrate skeleton requires a comprehensive view of the diverse skeletal cell types found in distinct developmental contexts, tissues, and species.To date, our knowledge of the molecular nature of the shark calcified extracellular matrix, and its relationships with osteichthyan skeletal tissues, remain scarce.Finally, we uncover a striking parallel, from a molecular and histological perspective, between the vertebral cartilage calcification of both species and discuss the evolutionary origin of endochondral ossification.

View Article: PubMed Central - PubMed

Affiliation: Institut des Sciences de l'Evolution de Montpellier, UMR5554, Université Montpellier, Centre National de la Recherche Scientifique, IRD, EPHE Montpellier, France.

ABSTRACT
Understanding the evolutionary emergence and subsequent diversification of the vertebrate skeleton requires a comprehensive view of the diverse skeletal cell types found in distinct developmental contexts, tissues, and species. To date, our knowledge of the molecular nature of the shark calcified extracellular matrix, and its relationships with osteichthyan skeletal tissues, remain scarce. Here, based on specific combinations of expression patterns of the Col1a1, Col1a2, and Col2a1 fibrillar collagen genes, we compare the molecular footprint of endoskeletal elements from the chondrichthyan Scyliorhinus canicula and the tetrapod Xenopus tropicalis. We find that, depending on the anatomical location, Scyliorhinus skeletal calcification is associated to cell types expressing different subsets of fibrillar collagen genes, such as high levels of Col1a1 and Col1a2 in the neural arches, high levels of Col2a1 in the tesserae, or associated to a drastic Col2a1 downregulation in the centrum. We detect low Col2a1 levels in Xenopus osteoblasts, thereby revealing that the osteoblastic expression of this gene was significantly reduced in the tetrapod lineage. Finally, we uncover a striking parallel, from a molecular and histological perspective, between the vertebral cartilage calcification of both species and discuss the evolutionary origin of endochondral ossification.

No MeSH data available.


Related in: MedlinePlus