Bone geometrical parameters. (A) Representative 3-dimensional (3D) models of WT and untreated and treated Chi/+ fish. Scale bar: 100 μm. (B) Micro-CT (μCT) analysis of bone volume (BV), vertebral thickness (V.Th), and BMD in WT, Chi/+ (i), and Chi/+ untreated and treated with 4PBA, ALN, and 4PBA + ALN (ii). (C) Representative 3D models of WT and untreated and treated p3h1^−/− fish. Scale bar: 100 μm. (D) μCT analysis of BV, V.Th, and BMD in WT, p3h1^−/− (i), and p3h1^−/− untreated and treated with 4PBA, ALN, and 4PBA + ALN (ii). In the graphs, each dot represents a single value. ^*p < .05, ^**p < .01, ^***p < .001. Abbreviations: ALN, alendronate; 4PBA, 4-phenylbutyrate.

Two-dimensional (2D) analysis of vertebral thickness and intervertebral disk (IVD) structure in Chi/+ and p3h1^−/− zebrafish. (A, B) 2D vertebral thickness (V.Th) distribution along the z-axis of WT and untreated mutants (i) and between untreated and treated mutants (ii). Dashed lines outline the vertebral centrum and abdominal (aEP) or caudal (cEP) endplates. (C) IVD structure drawing displaying vertebral endplate elements and toluidine blue–stained sections showing WT and treated and untreated Chi/+. Scale bar: 40 μm. (D) IVD structure qualitative evaluation in WT, Chi/+ (i), and chi/+ untreated and treated with 4PBA, ALN, and 4PBA + ALN (ii). In the graph, the percentage of normal, enlarged, or dislocated IVDs on total number of analyzed IVDs is reported (^*p < .05, ^**p < .01, ^****p < .0001). (E, F) 2D landmark scatterplot of WT and untreated mutant vertebrae (i) and untreated and treated mutant vertebrae (ii). Each dot represents a single landmark. Abbreviations: ALN, alendronate; cf, collagen I fiber bundles; dc, dense collagen I fiber bundles; el, elastin layer; fb, fibroblasts; nc, notochord cells; ne, notochord epithelium; ns, notochord sheath; Ob, osteoblasts; ot, osteocytes; 4PBA, 4-phenylbutyrate.

Analysis of osteoclast activity in Chi/+ and p3h1^−/− zebrafish. (A, B) TRAP staining quantification on haemal and neural arches and autocentrum of WT and Chi/+ (i) and Chi/+ untreated and treated with 4PBA, ALN, and 4PBA + ALN (ii). (C) Representative images of TRAP-stained WT and Chi/+ caudal vertebrae. (D, E) TRAP staining quantification on arches and vertebrae of WT and p3h1^−/− (i) and p3h1^−/− untreated and treated with 4PBA, ALN, and 4PBA + ALN (ii). (F) Representative images of TRAP-stained WT and p3h1^−/− caudal vertebrae. Red arrows indicate TRAP signal on vertebral bodies. The yellow asterisks indicate the intervertebral disc. Scale bar: 50 μm. In the graphs, the percentage of TRAP+ vertebrae on total number of analyzed vertebrae is reported. ^*p < .05, ^**p < .01, ^***p < .001, ^****p < .0001. Abbreviations: ALN, alendronate; NC, notochord; TRAP, tartrate-resistant acid phosphatase; 4PBA, 4-phenylbutyrate.

Analysis of osteoblast homeostasis and extracellular collagen maturation. (A) RT-qPCR analysis of bglap expression in WT, Chi/+ (i), and untreated and treated Chi/+ (ii). (B) RT-qPCR analysis of col1a1a expression in WT, Chi/+, and untreated and treated Chi/+. (C, D) Quantification of the immature (green-yellow) or mature (red) collagen fibers on total collagen content in WT, Chi/+ (i) and untreated and treated Chi/+ (ii) caudal vertebrae. (E) Representative images of picrosirius red–stained caudal vertebrae sections of WT and untreated or treated Chi/+. Scale bar: 100 μm. (F) RT-qPCR analysis of bglap expression in WT, p3h1^−/− (i), and untreated and treated p3h1^−/− (ii). (G) RT-qPCR analysis of col1a1a expression in WT, p3h1^−/− (i), and untreated and treated p3h1^−/− (ii). (H, I) Quantification of the immature (green-yellow) or mature (red) collagen fibers on total collagen content in WT, p3h1^−/− (i), and untreated and treated p3h1^−/− caudal vertebrae (ii). (J) Representative images of picrosirius red–stained caudal vertebrae sections of WT and untreated and treated p3h1^−/−. Scale bar: 100 μm. (K) Transmission electron microscopy representative images of Chi/+ osteoblasts and quantification of ER cisternae width in Chi/+ and Chi/+ treated with 4PBA + ALN. Scale bar = 500 nm. (L) Transmission electron microscopy representative images of p3h1^−/− osteoblasts and quantification of ER cisternae width in p3h1^−/− and p3h1^−/− treated with 4PBA + ALN. Scale bar = 500 nm. (Red arrowheads indicate ER cisternae transverse sections, yellow arrowheads indicate ER cisternae sagittal sections, pink asterisks indicate osteoblasts nuclei.). ^*p < .05, ^**p < .01, ^***p < .001, ^****p < .0001. Abbreviations: ALN, alendronate; ER, endoplasmic reticulum; RT-qPCR, reverse transcriptase quantitative PCR; 4PBA, 4-phenylbutyrate.

Analysis of osteocyte number and viability in Chi/+ and p3h1^−/−. (A) Osteocyte number normalized to vertebral area (Ot./vb.Ar.) performed on toluidine blue sections of WT, Chi/+ (i), and untreated and treated Chi/+ (ii). (B) TUNEL+ osteocytes on total osteocytes number (TUNEL(+) Ot./ total Ot. number) in WT, Chi/+ (i), and untreated and treated Chi/+ (ii). (C) Representative images of toluidine blue–stained vertebrae showing osteocytes (black arrows) in WT and untreated or treated Chi/+. Scale bar: 50 μm. (D) Representative images of TUNEL assay performed on WT and treated and untreated Chi/+ vertebrae. Red arrows indicate apoptotic osteocytes; nuclei are stained with DAPI (4′,6-diamidino-2-phenylindole). Scale bar: 100 μm. (E) Osteocyte number on vertebral area (Ot./vb.Ar.) performed on toluidine blue–stained sections of WT, p3h1−/− (i), and untreated and treated p3h1^−/ (ii). (F) TUNEL+ osteocytes on total osteocyte number (TUNEL(+) Ot./ total Ot. Number) in WT p3h1^−/− (i) and untreated and treated p3h1^−/− (ii). (G) Representative images of toluidine blue–stained vertebrae showing osteocytes (black arrows) in WT and untreated or treated p3h1^−/−. Scale bar: 50 μm. (H) Representative images of TUNEL assay performed on WT and treated and untreated p3h1^−/− vertebrae. Red arrows indicate apoptotic osteocytes; nuclei are stained with DAPI. Scale bar: 100 μm. In the graphs, each dot represents a single value. ^*p < .05, ^**p < .01, ^***p < .001, ^****p < .0001. Abbreviations: ALN, alendronate; NC, notochord; 4PBA, 4-phenylbutyrate.