Parallel Evolution of Ameloblastic scpp Genes in Bony and Cartilaginous Vertebrates

Abstract

In bony vertebrates, skeletal mineralization relies on the secretory calcium-binding phosphoproteins (Scpp) family whose members are acidic extracellular proteins posttranslationally regulated by the Fam20°C kinase. As scpp genes are absent from the elephant shark genome, they are currently thought to be specific to bony fishes (osteichthyans). Here, we report a scpp gene present in elasmobranchs (sharks and rays) that evolved from local tandem duplication of sparc-L 5' exons and show that both genes experienced recent gene conversion in sharks. The elasmobranch scpp is remarkably similar to the osteichthyan scpp members as they share syntenic and gene structure features, code for a conserved signal peptide, tyrosine-rich and aspartate/glutamate-rich regions, and harbor putative Fam20°C phosphorylation sites. In addition, the catshark scpp is coexpressed with sparc-L and fam20°C in tooth and scale ameloblasts, similarly to some osteichthyan scpp genes. Despite these strong similarities, molecular clock and phylogenetic data demonstrate that the elasmobranch scpp gene originated independently from the osteichthyan scpp gene family. Our study reveals convergent events at the sparc-L locus in the two sister clades of jawed vertebrates, leading to parallel diversification of the skeletal biomineralization toolkit. The molecular evolution of sparc-L and its coexpression with fam20°C in catshark ameloblasts provides a unifying genetic basis that suggests that all convergent scpp duplicates inherited similar features from their sparc-L precursor. This conclusion supports a single origin for the hypermineralized outer odontode layer as produced by an ancestral developmental process performed by Sparc-L, implying the homology of the enamel and enameloid tissues in all vertebrates.

Keywords: Scyliorhinus canicula; fam20°C; scpp; sparc-L; ameloblasts; enamel; enameloid; gene conversion; genomic convergence; jawed vertebrate evolution.

Fig. 1.

Characterization of a novel scpp gene in shark and ray genomes. Synteny and intron–exon structure of the sparc-L and scpp loci for the indicated osteichthyan (Lepisosteus oculatus) and chondrichthyan species (the holocephalan Callorhinchus milii; the shark S. canicula; the ray A. radiata). For synteny, triangles show relative gene position and orientation. The osteichthyan scpp members are represented as whole P/Q-rich or acidic clusters and not individually. For each exon, a color code legend identifies the UTRs or the encoded protein domains. Numbers at the bottom right corner of each exon indicate the position of the last encoded amino-acid, and numbers between two consecutive coding exons indicate the translation phase. Putative Fam20°C phosphorylation sites (SxE) are shown along each sequence. All exons are drawn to scale, except for the last exon which is trimmed. The position of the sparc-L and scpp in situ hybridization probes is indicated.

Fig. 2.

The lesser spotted catshark scpp gene is coexpressed with sparc-L and fam20°C in tooth and scale ameloblasts. Sections were performed at the level of developing scales (A–D, 8 cm-long embryos) and teeth (E–H, 9.5 cm-long embryos). Hematoxylin-Eosin-Safran histological staining (A and E) shows cells in the epithelium (ameloblasts, am) and the mesenchyme (odontoblasts, od), as well as the mineralized extracellular matrix (m). White arrowheads indicate secretory ameloblasts (sec am) and black arrowheads indicate maturation-stage ameloblasts (mat am). In situ hybridization signal identifies cells expressing scpp (B and F), sparc-L (C and G), or fam20°C (D and H).

Fig. 3.

Evolution of scpp from the sparc-L locus. (A) The cladograms represent three possible evolutionary scenarios: scpp genes in bony and cartilaginous fishes are orthologous and evolved from a single scpp duplication in all jawed vertebrates, followed by a scpp loss in holocephalans (Hypothesis 1); parallel duplication events generated scpp sequences in bony fishes and in cartilaginous fishes, followed by a scpp loss in holocephalans (Hypothesis 2); parallel duplication events generated scpp sequences in bony and elasmobranch fishes (Hypothesis 3). (B) Phylogenetic reconstruction of chondrichthyan Sparc-L and Scpp proteins. The phylogeny was performed with full-length Scpp and Sparc-L sequences and was rooted with the sea lamprey Sparc-L sequence (refer to supplementary material S14, Supplementary Material online for species names and accession numbers). The evolutionary model used was JTT + I + G4 and the alignment was 538 amino acid long (supplementary material S3, Supplementary Material online). Node support was evaluated with sh-lrt and 5000 ultra-fast bootstrap replicates, values are indicated on each node. Scpp proteins are shown in orange and red dots indicate bootstraps values superior to 95.

Fig. 4.

The elasmobranch scpp gene experienced nonallelic gene conversion with sparc-L specifically in selachimorphs. (A) Dot blots representing the position of highly conserved sequences between genomic regions containing sparc-L and scpp of S. canicula (left panel) and A. radiata (right panel). The shaded areas correspond to the conversion zones (aligned in supplementary material S7, Supplementary Material online). The 1 kb scale applies horizontally and vertically. (B) Unrooted phylogenetic tree performed with protein regions under gene conversion, under the JTT model (alignment from supplementary material S7, Supplementary Material online). (C) Unrooted phylogenetic tree performed with sequences excluding the conversion regions, the variable repeat and the Kazal/calcium-binding domains, under the FLU + F + G4 model (alignment from supplementary material S8, Supplementary Material online). In (B) and (C), Sparc-L and Scpp proteins are shown in grey and orange, respectively, bootstrap values are indicated on branches and red nodes indicate values superior to 95.

Fig. 5.

Evolutionary scenario for the turnover of the jawed vertebrate ameloblastic genetic toolkit. The composition of the enamel/enameloid mineralization toolkit is shown for the hypothetical stem jawed vertebrates (A), holocephalans (B), elasmobranchs (C), and osteichthyans (D), whose odontodes contain a dentine core (Dn) covered by an enamel/enameloid layer (En). Arrows indicate the phosphorylation (P) of Sparc-L (orange) and/or Scpp (red) proteins by Fam20°C (yellow). Sparc-L harbors a C-terminal Kazal/calcium-binding domain (Ca2+) absent from Scpp proteins. The holocephalan Sparc-L N-terminal domain contains 11 tandem duplications of an acidic motif (vertical lines) enriched in SxE sites. A cladogram shows for each jawed vertebrate lineage the ameloblastic expression of sparc-L and scpp genes (orange and red branches, respectively). Asterisks indicate parallel scpp duplication events. According to this model the ancestral enamel/enameloid mineralization toolkit was based on Sparc-L, a situation maintained in holocephalans. The elasmobranch toolkit includes Sparc-L and Scpp, whereas Sparc-L1 and Sparc-L2 were functionally replaced by the Scpp family in osteichthyans.