Skeletal Mineralization in Association with Type X Collagen Expression Is an Ancestral Feature for Jawed Vertebrates

Abstract

In order to characterize the molecular bases of mineralizing cell evolution, we targeted type X collagen, a nonfibrillar network forming collagen encoded by the Col10a1 gene. It is involved in the process of endochondral ossification in ray-finned fishes and tetrapods (Osteichthyes), but until now unknown in cartilaginous fishes (Chondrichthyes). We show that holocephalans and elasmobranchs have respectively five and six tandemly duplicated Col10a1 gene copies that display conserved genomic synteny with osteichthyan Col10a1 genes. All Col10a1 genes in the catshark Scyliorhinus canicula are expressed in ameloblasts and/or odontoblasts of teeth and scales, during the stages of extracellular matrix protein secretion and mineralization. Only one duplicate is expressed in the endoskeletal (vertebral) mineralizing tissues. We also show that the expression of type X collagen is present in teeth of two osteichthyans, the zebrafish Danio rerio and the western clawed frog Xenopus tropicalis, indicating an ancestral jawed vertebrate involvement of type X collagen in odontode formation. Our findings push the origin of Col10a1 gene prior to the divergence of osteichthyans and chondrichthyans, and demonstrate its ancestral association with mineralization of both the odontode skeleton and the endoskeleton.

Keywords: chondrichthyan; mineralization; scales; teeth; type X collagen.

Fig. 1.

Conserved synteny between Col10a1 genomic region in osteichthyan species: human, Xenopus, coelacanth, spotted gar, zebrafish; and region containing Col10a1 gene duplicates (numbered .1–.8) in chondrichthyan species: elephant shark, catshark, whale shark, and thorny skate. Genomic data for osteichthyan species were extracted from public databases (Ensembl, NCBI, XenBase) and analyzed to reconstruct the synteny blocks of duplicated Col10a1 genes in chondrichthyans. Distances are to scale within each species but may differ between species. Abbreviation: ex, exon.

Fig. 2.

Maximum likelihood phylogenetic tree based on analysis of Col10a1 nucleotide sequences with GTR+R4+F evolution model in IQ-TREE. Bipartition support was evaluated by 100 bootstrap replicates. Maximum support is shown by filled circles. Bootstrap supports below 75 are not displayed, and the branch thickness of their corresponding bipartition was decreased. Col8a1 and Col8a2 genes were used as outgroups and tree was rooted following Aldea et al. (2013). Predicted protein domains for catshark, elephant shark, and human Col10a1 sequences are displayed next to the gene names. Protein domains are labeled as SP—signal peptide, GXY—collagen triple alpha helix repeat, C1q—complement component 1q domain, and SE—serine-rich, acidic domain. Abbreviations: C., Chondrichthyes (cartilaginous fishes); O., Osteichthyes (bony fishes).

Fig. 3.

Histology and in situ hybridization on cryosections of catshark Scyliorhinus canicula skeletal tissues at stage 33. (A, B, C, V) Hematoxylin–Eosin–Saffron (HES) histological staining. (D–U) Expression of Col10a1 duplicates in developing teeth and scales of catshark detected with probes specific for each duplicate. Dotted lines indicate the mesenchymal compartment limits. Asterisk marks the mineralizing matrix, often seen as empty space due to decalcification. (W, X) Expression of Col10a1.4 duplicate in comparison to Col2a1 in mineralizing vertebral tissues of catshark. Black arrowhead in the neural arch points to the site of globular mineralization; open arrowhead points to the site of lamellar mineralization; black arrowhead in the vertebral body points to the site of fibrous mineralization. Scale bars: 50 µm (A–U), 200 µm (V–X). Abbreviations: mat. amel., maturation stage ameloblasts; M’s, Meckel’s cartilage; notoch, notochord; od., odontoblasts; sec. amel., secretory ameloblasts.

Fig. 4.

Expression of zebrafish col10a1a and col10a1b and Xenopus Col10a1 during tooth development detected with in situ hybridization. (A, B) col10a1a expression in whole-mount zebrafish larvae at 4 and 6 days post fertilization (dpf). (C) col10a1a expression in post in situ paraffin sections of zebrafish larvae at 5 dpf. (D, E) col10a1b expression in whole-mount zebrafish larvae at 4 and 6 dpf. (F) col10a1b expression in post in situ paraffin sections of zebrafish larvae at 4 dpf. (G) HE staining of a longitudinal section through a developing tooth bud of a Xenopus larva at stage NF57. (H, I) Col10a1 expression in Xenopus tooth bud on successive transversal sections. Open arrowheads point at developing pharyngeal teeth and asterisk marks the mineralizing matrix. Scale bars: 100 µm (A, B, D, E), 50 µm (C, F), 20 µm (G, H, I). Abbreviations: amel., ameloblasts; cl, cleithrum; d, dentary; m, maxilla; od., odontoblasts; op, opercle; p, parasphenoid; sec. amel., secretory ameloblasts.

Fig. 5.

Proposed scenario for Col10A1 locus evolution and subfunctionalization. Col10a1 genes with expression in dermal skeleton and/or endoskeleton are marked as green boxes, with expression in odontode skeleton are marked as blue boxes and with unknown expression are marked by gray boxes. Tandem duplication of Col10a1 genes in chondrichthyan ancestor is explained in detail in supplementary figure 2 (Supplementary Material online).