Enault S et al. (2015), Molecular footprinting of skeletal tissues in t...

XB-ART-51358

Front Genet 2015 Sep 15;6:283. doi: 10.3389/fgene.2015.00283.

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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 .

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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.

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Species referenced: Xenopus tropicalis
Genes referenced: col1a1 col1a2 col2a1 nlrp1
GO keywords: bone mineralization

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	Figure 1. Cartilage calcification and collagen expression in Scyliorhinus canicula radials and Meckel's cartilage. (A) Schematic drawing of the pectoral fin anatomy from 7 cm long S.c. embryos and of the orientation of the paraffin sections shown in C–G (blue dotted lines). Rostral and caudal refer to the embryonic axis. (B) General histology of pectoral skeletal elements, with the center of the cartilaginous element located at the bottom. (C,C') Alizarin red and Alcian blue double staining. (D–F) Gene expression patterns in the pectoral fin for Sc-Col1a1 (D), Sc-Col1a2 (E), and Sc-Col2a1 (F). (G) Immunofluorescence using an anti-Type II collagen (Col2) antibody specifically marks the pectoral fin cartilaginous condensations. (H) Schematic drawing of the jaw anatomy from 9 cm-long S.c. embryos (ventral view) and of the orientation of the paraffin sections shown in (J–N) (blue dotted line). (I) General histology of Meckel's cartilage, with the center of the cartilaginous element located at the top. The arrowheads in (I,J'–N') demarcate the fibrous perichondrium from the cartilage. (J) Alizarin red and Alcian blue double staining. (J') Higher magnification of a tesserae located in a similar region as the area boxed in (J) and stained with HES. (K–M) Gene expression patterns in the jaw for Sc-Col1a1 [the inset in (K) shows a Sc-Col1a1 positive dermal denticle from the same section], Sc-Col1a2 (L) and Sc-Col2a1 (M). (N) Immunofluorescence using an anti-Type II collagen (Col2) antibody specifically marks the cartilaginous condensations of Meckel's cartilage. Insets in (C–N) are shown at higher magnification in (C'–N'), respectively. CZ, calcification zone of the tesserae; Ch, chondroctyces; Fb, fibroblasts; Pc, perichondrium; Pq, palatoquadrate. Scale bars: (C–G), 250 μm; (J–N), 100 μm.
	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.
	Figure 3. Comparison of the Col1a1, Col1a2, and Col2a1 expression patterns during Xenopus tropicalis hindlimb development. Stage NF54 (top panel) or NF60 (bottom panel) hindlimbs were examined by whole mount Alizarin red staining (insets), sectioned along the proximo-distal axis and stained with HES, (A–C, M–O) or processed by in situ hybridization for the Xt-Col1a1, Xt-Col1a2, and Xt-Col2a1 probes, (D–L, P–X). Results are shown for the whole skeletal element (left column, scale bar: 500 μm) and higher magnifications of the diaphysis (middle column, scale bar: 50 μm) and epiphysis (right column, scale bar: 50 μm). Arrows and arrowheads show osteoblasts and osteocytes, respectively. In situ hybridization signal is light to dark blue, and brown endogenous X.t. pigment cells are visible on most sections. Legend: Bo, bone; Ch, chondrocytes; Me, medulla; Pe, perichondrium; Sm, striated muscles.
	Figure 4. Histology of the developing Xenopus tropicalis vertebrae. (A) Schematic drawing of transverse sections running through the lateral or the dorsal region of the neural arches. (B) Color code used to represent the distinct skeletal tissues of the X.t. vertebrae in (F,I,N,T,P,V). (C) Whole mount Alizarin red staining of stage NF54 vertebral column (lateral view, anterior to the left). (D–I) Histology of the stage NF54 vertebrae examined with HES (D,E,G,H). (J) Whole mount Alizarin red staining of stage NF57 vertebral columns (lateral view, anterior to the left). (K–V) Histology of the stage NF57 vertebrae examined with HES (K,O,Q,U), Alcian blue (L,R) and Alizarin red (M,S). Insets in D, G, K and Q are shown in F, I, P and V, respectively. Panels E, H, K, O, Q and U are schematized in F, I, N, P, T and V, respectively. Abbreviations: nt, neural tube; ntc, notochord. Scale bars: 1 mm in (C,J); 250 μm in (D,G); 50 μm in (E,F) and (H,I); 500 μm in (K–N) and (Q–T); and 50 μm in (O,P,U,V).
	Figure 5. Skeletal expression patterns the Xt-Col1a1, Xt-Col1a2, and Xt-Col2a1 during Xenopus tropicalis vertebrae development. Transverse sections of stage NF54 (A–F) and NF57 (G–L) vertebrae processed for in situ hybridizations using the Xt-Col1a1, Xt-Col1a2, and Xt-Col2a1 probes, as indicated. Black arrowheads show loose (A,B) or perichondral (D–F) cells. White arrowheads in (C,I) show Xt-Col2a1 positive epithelial non-vacuolated cells of the notochord. Arrows point at osteoblasts expressing Xt-Col1a1 (G,J), Xt-Col1a2 (H,K), or Xt-Col2a1 (I,L). In (I,L), calcified, Alizarin red-positive cartilaginous regions are marked by an asterisk and the dotted lines demarcates expression boundaries between Xt-Col2a1 positive and Xt-Col2a1negative chondrocytes. In situ hybridization signal is light to dark blue. Brown endogenous X.t. pigment cells are also visible in the vicinity of the dorsal neural arch (D–F, J–L). Scale bar in (A) represents 50 μm in (A–F); scale bar in (G) represents 50 μm in (G–L).
	Figure 6. An evolutionary scenario for bone formation and perichondral calcification in jawed vertebrates. Bone/perichondrium histology and gene expression patterns were mapped onto a simplified vertebrate phylogenetic tree to deduce ancestral states and polarize evolutionary change. We propose that the ancestral Clade A fibrillar collagen gene (i.e., before the duplications that produced the distinct member of this family) was expressed in the non-calcified perichondrium. This expression pattern was inherited by the unique cyclostome fibrillar collagen gene which is more closely related to the Col2a1 subgroup. In jawed vertebrates, perichondral cells and osteoblasts maintained high levels of Col1a1 and Col1a2 while the Col2a1 osteoblastic expression was dramatically reduced in most (but not all) lineages. The presence of bone in placoderms and tetrapods supports the idea that the calcified fibrous perichondrium observed in some chondrichthyan species either represents bone evolutionary remnants (Hypothesis 1) or a secondary gain of calcification (Hypothesis 2). Osteocytes have been omitted for the sake of simplicity. See text for details.
	Figure 7. An evolutionary scenario for cartilage calcification in jawed vertebrates. Expression patterns and cartilage matrix calcification were mapped onto a simplified vertebrate phylogenetic tree to deduce ancestral states and polarize evolutionary change. We propose that, in the last vertebrate common ancestor, the expression of Col2a1 experienced a strong downregulation in maturing, non-calcified, cartilaginous regions. This downregulation was subsequently inherited by distinct vertebrate lineages, and is associated to hard cartilage in cyclostomes and to calcified cartilage in jawed vertebrates. The chondrichthyan and osteichthyan representatives analyzed in this study display a calcified Col2a1-negative vertebral cartilage, a likely jawed vertebrate synapomorphy. Tesserae calcification, a recent chondrichthyan innovation, occurs in the absence of Col2a1 downregulation. Perichondrium and bone have been omitted for the sake of simplicity. See text for details.

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