Bone is the most widespread mineralized tissue in vertebrates, and its formation represents a major leap in vertebrate evolution. Although chondrichthyans produce dermal bone (for example teeth, dermal denticles and fin spines) and calcified cartilage5,21, unlike bony vertebrates, their cartilage is not replaced with endochondral bone. Among vertebrates, the earliest mineralized tissue was found in the feeding apparatus of extinct jawless fishes, the conodonts21. Early dermal bone was found in extinct jawless vertebrates such as heterostracans, whereas perichondral bone surrounding the cartilage was found in several fossil jawless vertebrates (osteostracans, galeaspids) and jawed vertebrates5 (placoderms, acanthodians; Fig. 3). However, the highly complex process of endochondral ossification is unique to bony vertebrates. The C. milii genome sequence provided a unique opportunity to address the question of why the endoskeleton of chondrichthyans is not ossified.

Figure 3: Genetic events underlying the emergence of bone formation in vertebrates. Duplication of Sparc by whole-genome duplication initially gave rise to Sparcl1, and the subsequent tandem duplication of Sparcl1 gave rise to the SCPP gene family responsible for endochondral ossification. Because the sea lamprey genome contains only Sparc but no Sparcl1 (ref. 22), we have placed the genome duplication event that gave rise to Sparcl1 after the divergence of jawless vertebrates from the jawed vertebrate ancestor. The sister relationship of chondrichthyans and acanthodians is based on ref. 37. SCPP, secretory calcium-binding phosphoprotein gene family member. †, extinct. PowerPoint slide Full size image

We searched the C. milii genome assembly and transcriptomes for genes known to be involved in bone formation in osteichthyans (Supplementary Note X). All gene family members involved in bone formation were present, except the secretory calcium-binding phosphoprotein (SCPP) gene family (Supplementary Table X.1). This gene family encodes a diverse array of secreted phosphoproteins that arose from the gene Sparc-like 1 (Sparcl1) through tandem duplication, and Sparcl1 itself arose from an ancient metazoan gene, Sparc, through whole-genome duplication22. There are two main categories of SCPP genes: one group encodes acidic proteins and the other encodes proline- and glutamine-rich (P/Q-rich) proteins. In the human genome, the two groups are found in two different clusters on chromosome 4; the acidic SCPP genes (SPP1, MEPE, IBSP, DMP1 and DSPP, collectively known as SIBLING genes) occur between PKD2 and SPARCL1, whereas the P/Q-rich SCPP genes are found in the enamel matrix protein-SCPP cluster ∼17 megabases downstream of SPARCL1 (Supplementary Fig. X.2). Acidic SCPP or SIBLING genes are involved in the ossification of collagenous matrix in bone and dentine, and P/Q-rich SCPP genes are involved in the production of enamel, milk, tears and saliva. Although there are variable numbers of P/Q-rich SCPP genes in teleosts23, there is a single SIBLING gene, spp1, in zebrafish and medaka. Zebrafish spp1 (also known as osteopontin) is expressed specifically in osteoblasts24 and has therefore been proposed to have a primary function in bone formation similar to its mammalian orthologue23.

The C. milii genome contains both Sparc and Sparcl1 on different scaffolds that show extensive conserved synteny with orthologous loci in human and other bony vertebrates (Supplementary Figs X.1 and X.2). However, there is no SCPP gene cluster in the intergenic region between Pkd2 and Sparcl1 or elsewhere in the genome (Supplementary Fig. X.2). The genomic and transcriptomic resources available for other cartilaginous fishes such as the little skate (Leucoraja erinacea) and the small-spotted catshark (Scyliorhinus canicula) as well as the genome assembly of the sea lamprey, a jawless vertebrate, also do not contain any SCPP genes (Supplementary Note X). These findings suggest that the tandem duplication of Sparcl1 that gave rise to SCPP genes occurred in the common ancestor of osteichthyans after this lineage split from the chondrichthyan lineage (Fig. 3). Because SCPP genes have a crucial role in the formation of bone, we propose that their absence from C. milii explains the absence of bone from the endoskeleton of cartilaginous fishes.

To test this hypothesis, we used two different methods to disrupt the function of the single bone-specific SIBLING gene spp1 in zebrafish. The knockdown of spp1 using two gene-specific morpholinos (ATG MO and E2-I2 MO) resulted in a significant reduction in endochondral and dermal bone formation by comparison with embryos injected with 5-base-pair-mismatch control morpholinos (Supplementary Fig. X.4). Unlike the transient effects exerted by morpholinos, the genetic interference afforded by the CRISPR/Cas9 system25 results in heritable genomic modifications; indeed, targeting exons 6 and 7 of the spp1 gene using the CRISPR/Cas9 system resulted in the generation of specific insertion/deletion mutations at the target sites, including a ∼2.6-kilobase deletion when exons 6 and 7 were simultaneously targeted (Supplementary Fig. X.6). Embryos 5 days post-fertilization (dpf) with deletions in exon 7 alone or in both exon 6 and exon 7 of spp1 showed a significant reduction in the formation of endochondral and dermal bone (Fig. 4), with the defect in bone formation persisting in 15-dpf mutant embryos (Supplementary Fig. X.9). The similar phenotypes obtained using two different methods of manipulation indicate that the effects on bone formation are specific, and strongly suggest that spp1 has an essential role in the modulation of bone formation in zebrafish.

Figure 4: Targeted mutagenesis of zebrafish spp1 by sgRNA:Cas9 results in reduced bone formation. a, spp1 is specifically expressed in cells surrounding the bone matrix. Ventral view of a 5-dpf embryo hybridized with a spp1-specific RNA probe. Yellow labels, endochondral bones (cb5, ceratobranchial 5; ch, ceratohyal); white labels, dermal bones (bsr, branchiostegal ray; cl, cleithrum; d, dentary; en, entopterygoid; op, operculum; ps, parasphenoid). b, Ventral view of a 5-dpf wild-type (WT) embryo stained with alizarin red to reveal sites of bone deposition (red fluorescence). mx, maxilla. c, Bright-field image merged with b to visualize anatomical structures and locations of bone deposition simultaneously. d, Ventral views of 5-dpf embryos injected with Cas9 mRNA together with single guide RNA (sgRNA) targeting spp1 exon 6, exon 7 or both (alizarin red staining). The embryos were scored as normal (resembling wild type), mild or strong bone phenotypes, with the latter showing the greatest reduction in bone formation. The variations in the extent of bone reduction are presumably due to somatic chimaerism with regard to spp1 disruption. e, Proportions of mild and strong bone phenotypes induced by disruption of spp1 by sgRNA/Cas9. Targeting of exon 7 (n = 206 embryos) or both exons 6 and 7 (n = 143) resulted in significantly higher proportions of strong bone phenotype (P < 0.01, Fisher’s exact test) compared with control injections of Cas9 mRNA (n = 190) and exon 7 sgRNA (n = 143) (Ex6, Cas9: n = 72). PowerPoint slide Full size image

The results of the zebrafish spp1 knockdown experiments provide strong support for our hypothesis that the absence of SCPP genes from cartilaginous fishes is related to the unossified nature of their endoskeleton. In turn, the absence of SCPP genes from chondrichthyans raises questions about the genetic basis of dermal and perichondral bone formation in chondrichthyans, placoderms, acanthodians and extinct jawless vertebrates. We speculate that one or more SCPP-related genes, probably Sparc, Sparcl1 or both, mediate the mineralization of skeleton in these vertebrates.