<i>wnt16</i> is expressed in dermomyotome-like cells at 22 hpf.

(A) A schematic of a transverse section through the zebrafish trunk with compartments labeled and color coded. (B-C) Chromogenic in situ hybridization of an anterior (note the yolk in the ventral space) transverse section through the zebrafish trunk shows cells expressing wnt16 and pax7a are located in the external cell layer (magnified in C). (D) Transverse sections, beginning in the anterior trunk (left), and moving posteriorly (right), representing less mature somites, show variation in wnt16+ labeling.</p

<i>WNT16</i> is a gene of major effect on lean mass at the <i>CPED1-WNT16</i> locus.

(A) Schematic depicting variant associations and all genes with transcriptional start sites within ±500kb of the most significantly associated SNP at 7q31.31. (B) Z-scores for somatic mutants for tspan12, ing3, cped1, wnt16, and fam3c. (C) Segmentation of microCT images for cped1w1003 mutants for bone (top) and lean (bottom) tissue. (D) Z-scores for cped1w1003 and wnt16w1001 mutants. P-values were determined using an unpaired t-test with the number of fish per group provided in the figure. *p<0.05, **p<0.01, ***p<0.001.</p

<i>wnt16</i> suppresses myotome expansion and promotes notochord radial expansion.

(A) Transverse views of phalloidin-stained 3dpf animals. em: epaxial myotome, hm: hypaxial myotome, ms: myosepta, nc: notochord, nt: neural tube, t: trunk. (B-I) Quantification of myotome (B-F) and notochord (G-I) morphology. P-values were determined using an unpaired t-test. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns: not significant.</p

Isolation of <i>wnt16</i> mutant alleles.

(A) Sequence and genomic location of w1001, w1008, and w1009. Grey highlight indicates gRNA target sequence used for CRISPR-based gene editing with PAM underlined. (B) Predicted effects of alleles on amino acid sequence. (C) RT-PCR assessing wnt16 transcript in wnt16w1001 mutants. No evidence of transcript reduction or alternative splicing is observed. (D) Calcein staining of 13 dpf animals show similar reductions in vertebral mineralization and post-cranial body length in w1001, w1008, and w1009 mutants. (E) Quantification of mineralized area shows similar changes in mutants for all three alleles. (TIF)</p

Zygotic and maternal zygotic <i>wnt16</i>^<i>-/-</i> mutant phenotypes.

P-values were determined using a two-way ANOVA with Fisher’s LSD post hoc test. *p (TIF)</p

Adult <i>wnt16</i>^<i>-/-</i> mutants exhibit altered lean mass and morphology.

(A) Segmentation of microCT images for bone (top) and lean (bottom) tissue. Shown are average intensity projections. (B) wnt16-/- mutants exhibit reduced fineness ratio. Fish images adapted from [46]. (C) Lean cross-sectional area as a function of distance along the anteroposterior axis. Thickness of the line indicates standard error of the mean (n = 4-6/group). Arrowhead indicates approximate boundary between the anterior and posterior swim bladders. (D) Lean volume in the trunk, anterior trunk, and posterior trunk. wnt16-/- mutants exhibit reduced lean volume in the posterior trunk compared to controls (wnt16+/+ and wnt16+/-) (E) wnt16-/- mutants exhibit reduced swim bladder length in the posterior, but not anterior, chamber compared to wildtype. (F) High-resolution contrast-enhanced microCT reveals altered myomere width and angle in wnt16-/- mutants as indicated by dotted lines in the top part of the myotome compartment in volumetric renderings. P-values were determined using an unpaired t-test (B) or two-way repeated measures ANOVA (D and E) with Fisher’s LSD post hoc test. *pFig 1 from the following paper: Walker, J.A., Alfaro, M.E., Noble, M.M., and Fulton, C.J. (2013). Body fineness ratio as a predictor of maximum prolonged-swimming speed in coral reef fish. PLoS One 8, e75422. The paper of Walker et al., which was published in PLoS One, applies the Creative Commons Attribution 4.0 International (CC BY) license (https://journals.plos.org/plosone/s/licenses-and-copyright).</p

RNA ISH for markers of early muscle (<i>myog</i>, <i>pax7a</i>) and notochord (<i>ntla/tbxta</i>) differentiation in <i>wnt16</i>^-/- mutant embryos at 1 dpf.

Scale bar: 200 μm. (TIF)</p

Inference of myomere morphology from vertebral measures.

(A) Schematic demonstrating correlated features. (B-D) wnt16-/- mutants exhibit altered centrum length and neural arch angle. P-values were determined using an unpaired t-test. **p (TIF)</p

Data used for calculating Z-scores for Fig 9D.

(TIF)</p

<i>wnt16</i> is necessary for notochord sheath mineralization.

(A) Calcein staining in wnt16-/- larvae (WT: 5.7mm SL). In wnt16-/- mutants, mineralized domains are irregular in shape, incompletely proceeding to the dorsal surface from the ventral surface of the notochord. (B-G) Quantification of mineralizing domain morphology. In (C-G), each point represents a single fish, with values averaged from vertebrae 1–16. (H) Calcein staining in wnt16-/- larvae (WT: 7.0mm SL). (I) Dual fluorochrome staining shows trapezoid-like mineralized domains in wnt16-/- mutants eventually became rectangle-like. (J-L) Quantification of centrum length (J), standard length (K), and cranial length (L). (M) Visualization of osteoblastic cells using Tg[sp7:EGFP]. EGFP domains in mutants appear trapezoid-like with reduced expression on the dorsal surface, resembling calcein domains in (A). (N) Visualization of osteoclastic cells using Tg[ctsk:DsRed]. Similar DsRed domains are observed in wildtype and wnt16-/- mutants. P-values were determined using one-way ANOVA with Fisher’s LSD post hoc test. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns: not significant.</p