Fig. 1

The Cys-His-Gly triplet within the WNT motif is necessary for Wnt16 function: A. (Top) Sequence alignment of Xenopus (X) Wnt8, human (H) WNT1-16, and zebrafish (D) Wnt16. Asterisks indicate the highly conserved Cys-His-Gly triplet within the WNT motif. "A" indicates the serine that is post-translationally acylated. (Bottom) Darker and taller bars indicate higher consensus at that amino acid position. B. Sequence and genomic location of wnt16w1012. Grey highlight indicates gRNA target sequence in exon 4 used for CRISPR-based gene editing. C. Predicted effects of allele on amino acid sequence with location on protein indicated. D. 2-way ANOVA analyses of volume, tissue mineral density (TMD), and thickness for centrum, haemal arch, and neural arch, and centrum length. n=10/group for wnt16w1001 clutchmate controls and wnt16w1012 homozygous mutants and clutchmate controls. n=6/group for wnt16w1001 homozygous mutants. Purple highlight and table indicate phenotypic features of wnt16w1001 and wnt16w1012 that differ significantly from controls. E. Maximum intensity projections of representative microCT scans for one wnt16w1012 fish and one clutchmate control. F. (Top) Structural model of the wild-type Wnt16 protein (green) with inset showing superimposition of the protein encoded by wnt16w1012 (orange). Blue dotted lines represent hydrogen bonds. “CHG” represents the location of the Cys-His-Gly triplet on Wnt16. Arrows indicate the location of acyl addition (*Ser215 is equivalent to Ser218 but shifted due to triplet deletion). (Bottom) Tables showing missing/created hydrogen bonds within the thumb of the encoded protein of wnt16w1012. Numbers in parentheses indicate the number of those specific bonds that are missing or created.