Venkatesh et al., 2014 - Elephant shark genome provides unique insights into gnathostome evolution. Nature   505(7482):174-179 Full text @ Nature

Fig. 4

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, ceratobranchial5; 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 exon6, exon7 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 exon7 (n = 206 embryos) or both exons6 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 exon7 sgRNA (n = 143) (Ex6, Cas9: n = 72).

Fig. SX.3

Zebrafish spp1 is expressed from 2 dpf specifically in cells surrounding the bone matrix.

Lateral and ventral views of 2 to 5 dpf embryos hybridized with spp1 RNA probe. There was no expression at 1 dpf. Endochondral bones (ch, ceratohyal; cb5, ceratobranchial 5) are highlighted in yellow whereas dermal bones (bsr, branchiostegal ray; cl, cleithrum; d, dentary; en, entopterygoid; mx, maxilla; op, operculum; ps, parasphenoid) are highlighted in white.

Fig. SX.4

Reduction of bone deposition in spp1 zebrafish morphants.

(a) spp1 is specifically expressed in cells surrounding the bone matrix. Ventral view of a 5 dpf embryo hybridized with spp1 RNA probe. Endochondral bones (ch, ceratohyal; cb5, ceratobranchial 5) are highlighted in yellow whereas dermal bones (bsr, branchiostegal ray; cl, cleithrum; d, dentary; en, entopterygoid; op, operculum; ps, parasphenoid) are highlighted in white. (b) Ventral views of 5 dpf wild type, ATG MO, ATG control MO, E2-I2 MO and E2-I2 control MO embryos. Embryos were stained with alizarin red to reveal sites of bone deposition and imaged under red fluorescence. For one wild type embryo (bottom left), a merged bright field image and red fluorescence signal is shown to simultaneously visualize anatomical structures and bone deposition. (c) Proportion of embryos showing normal phenotype (resembling wild type), ‘mild’ bone-phenotype and ‘strong’ bone-phenotype (the latter showing the most reduction of bones). ATG MO and E2-I2 MO morphants show a significantly higher proportion of strong bone-phenotype (p<0.01, Fisher’s exact test)compared to their respective controls. (d) Bright field images of embryos shown in panel (b) showing normal growth of morphant embryos.

Fig. SX.5

spp1 gene knockdown in zebrafish.

(a) Five dpf zebrafish embryos stained with alcian blue for cartilage. Wild type, ATG morphant and E2-I2 morphant embryos are shown. Morphant embryos do not show any discernible change in cartilage matrix formation. (b) E2-I2 MO inhibits splicing of spp1 in zebrafish. Upper panel shows the position of RT-PCR primers (P1, P2 and P3) in relation to spp1 gene. Lower panel shows the RT-PCR fragments separated on a 3% agarose gel. The wild type spliced product is represented by a 120 bp fragment, whereas unspliced products are represented by the 87 bp and 230 bp fragments. Splicing of intron 2 is considerably inhibited in 4-dpf and 5-dpf embryos injected with E2-I2 MO, while normal splicing occurs in 4-dpf and 5-dpf embryos that are wild-type or injected with E2-I2 control MO.

Fig. SX.7

Brightfield images of embryos shown in Fig. 4d.

Embryos injected with Cas9 mRNA + various sgRNA showed an overall growth comparable to wild type.

Fig. SX.8

Alcian blue staining of embryos injected with Cas9 mRNA and various sgRNAs.

Although bone formation was reduced in embryos treated with Cas9 mRNA + sgRNAs, the cartilage development was unaffected.

Fig. SX.9

Targeted mutagenesis of zebrafish spp1 by sgRNA:Cas9 results in reduced bone formation in 15 dpf embryos.

Left panel: ventral view of a 15 dpf wild type embryo stained with Alizarin red to reveal sites of bone deposition (red fluorescence). Its light microscopy image is given below. Endochondral bones (cb5, ceratobranchial 5; ch, ceratohyal; hm, hyomandibula; mpt, metapterygoid; q, quadrate) are highlighted in yellow whereas dermal bones (bh, basihyal; bsr, branchiostegal ray; cl, cleithrum; d, dentary; en, entopterygoid; mx, maxilla; op, operculum; ps, parasphenoid) are highlighted in white. Entopterygoid (en) and metapterygoid (mpt) are fused and hence cannot be readily distinguished. However, both are affected in spp1 mutants shown in the right panel. Right panel: ventral views of 15 dpf embryos injected with Cas9 mRNA together with single guide RNA (sgRNA) targeting spp1 exon 6, exon 7 or both exons. The ‘strong’ bone-reduction phenotype embryos are shown (stained with Alizarin red). Light microscopy images of the mutants show normal development of the embryos.

Acknowledgments:
ZFIN wishes to thank the journal Nature for permission to reproduce figures from this article. Please note that this material may be protected by copyright. Full text @ Nature