piezo1 and piezo2a are essential for bone formation and mineral deposition in early stage of development. (A,B) Gross phenotypes and (C,D) mineralization patterns of sibling and piezo1^−/−; piezo2a^−/− mutant at 8 days after fertilization (dpf). White arrow indicates uninflated swim bladder. Scale = 500 μm. (E,F) Magnified images of the anterior body of sibling and double mutant. Numbers 1 to 8 indicate the position of each vertebral bone. The dashed circle indicates swim bladder. Notably, the swim bladder in double mutant was small and uninflated. (G) Schematic diagram of generating piezo transient KO mutant (CRISPRant). Comparison of 3D reconstruction of micro-CT images of vertebral bone at caudal part from (H) wildtype, (I)piezo1 sgRNA injected to piezo2a^−/−, and (J)piezo2a sgRNA injected to piezo1^−/− at 2.5 months after fertilization.

Generating piezo1 in-frame mutant. (A) Schematic illustration of CRISPR target in piezo1 gene with a guide RNA targeting splice acceptor site of exon2. Through this guideRNA, the splice acceptor site was eliminated, generating cryptic acceptor site which deleted several bases of internal exon 2. (B) Confirmation of amino acid sequence. Quantification of relative mRNA level of (C)piezo1 and (D)piezo2a in piezo1^{11aa del/11aa del}. Values are presented as mean ± SD and analyzed using Student’s t-test. **p < 0.01. (E) Representative traces of negative pressure-induced inward currents recorded at −80 mV in negative control (NC), zebrafish Piezo1 Wildtype (WT), and zebrafish 11aa del-Piezo1. Blue arrows indicate the time when negative pressure was applied. Grey dashed lines indicate baseline of the current. (F) Measurement of current amplitude, measured in arbitrary units (AU). Values are presented as mean ± SD and analyzed using one-way ANOVA followed by Tukey’s test. ****p < 0.0001. ns indicates not significant.

Phenotype of piezo1 in-frame mutant (piezo1^{11aa del/11aa del}). (A) Time course observation of mineralized bone using alizarin red S staining between wildtype and piezo1^{11aa del/11aa del} mutant from larvae (8 dpf) to young adult (35 dpf) (Used scale 500 μm) and 3D rendering of micro-CT images of wildtype and piezo1^{11aa del/11aa del} mutant at 4 months. (B) Graph depicting body length. (C) Average of total Cobb angle. Cobb angles were measured according to previous study (Bearce et al., 2022). Values are presented as mean ± SD and analyzed using Student’s t-test. ****p < 0.0001. ns means non-significant.

Detailed analysis of vertebra shape. (A) Comparison of 3D reconstruction of micro-CT images of vertebral bone between wildtype and piezo1^{11aa del/11aa del} at the abdominal part from side and top view. (B) Schematic diagram and quantification of the (C) length ratio (L1/L2) and (D) height ratio (H1/H2) of single vertebrae measurement. About 6 vertebrae from wildtype (N = 5) and mutant (N = 5) were assessed. Length aspect ratio and height aspect ratio were significantly more variable in mutant (p < 0.0001, analyzed using F-test two samples for variances). (E) The micro-CT reconstructions of 3 months zebrafish from both the wildtype and piezo1^{11aa del/11aa del} mutant specimens were performed. The reconstructions were color-enhanced to represent bone density value. To minimize measurement error, trunk or abdominal part was chosen to measure TMD value due to low bone kinks, indicated as white boxes. (F) Measurement of Tissue Mineral Density (TMD). Values are presented as mean ± SD and analyzed using Student’s t-test. **p < 0.01.

Abnormal osteoblast function. (A,B) Representative image of alkaline phosphatase staining in vertebral bone section of wildtype (N = 4) and piezo1^{11aa del/11aa del} (N = 5). Red boxes indicate the magnified area of growth plate. Blue arrow indicates center of growth plate, orange arrow indicates growth plate at the opposite site. Scale bar is 100 μm. (C) Fluorescence images of vertebral bone of wildtype; (osx-GFP) and piezo1^{11aa del/11aa del}; (osx-GFP) at 25 dpf. (D) Quantitative analysis of fluorescence area. Values are presented as mean ± SD and analyzed using Student’s t-test. ***p < 0.001. mRNA level of(E) osterix, mid-stage osteoblast marker gene, and(F) osteoclacin/bglap, late-stage osteoblast marker gene. Values are presented as mean ± SD and analyzed using Student’s t-test. ****p < 0.0001.

Abnormal changes of vertebral bone in piezo1^{11aa del/11aa del}. (A) Normal feature of bone; (B) Intervertebral disc calcification (IC); (C) Bone fusion (BF); (D) Ectopic calcification (EC); (E) End-plate sclerosis (ES); (F) Osteophytes formation (OP). (G) Heat map depicting the position and frequency of abnormal features of bone between wildtype and piezo1^{11aa del/11aa del}.

Introducing functional piezo1 gene or increasing muscle mass can alleviate scoliosis symptoms. (A) Schematic diagram of rescue plasmid. Notably, sp7/osterix promoter was used to express functional piezo1 gene. (B) Comparison of 3D reconstruction of micro-CT images. (C) Quantification of TMD from 5 individual segments of abdominal vertebrae of wildtype (N = 6), piezo1^{11aa del/11aa del} mutant (N = 8), rescue mutant fish (N = 6), and double mutant of piezo1^{11aa del/11aa del}; mstnb^−/−(N = 5). (D) Combined Cobb angle of piezo1^{11aa del/11aa del} mutant (N = 8) rescue mutant fish (N = 7), and double mutant of piezo1^{11aa del/11aa del}; mstnb^−/− (N = 6). (E) Sagittal section of the spine. Rescue mutant and piezo1^{11aa del/11aa del}; mstnb^−/− have reduced the number of bone with IVD calcification (white arrows). (F) Graph depicting the percentage of bone with IVD calcification across bone segments 9 to 31. Values are presented as mean ± SD and analyzed using one-way ANOVA followed by Tukey’s test. ns = non-significant; **p < 0.01; ***p < 0.001; ****p < 0.0001.