Vertebral column deformities in Chi/+ and Chi/+_LP mutants. (A) Representative X-rays of three months old WT and Chi/+ (representative also for the malformations diagnosed in Chi/+_LP) zebrafish show severe vertebral column deformities in mutants, i.e. abdominal kyphosis (blue arrow), abdominal lordosis (white arrow), vertebral body compressions (white asterisks) and hemivertebra (black asterisk). (B) Frequency of malformations. Chi/+ mutants (n=20) show increased frequency of kyphosis, lordosis, scoliosis and vertebral body compressions in the abdominal and caudal vertebral column compared to WT animals (n=26). Chi/+_LP animals (n=18) display reduced kyphosis and lordosis of the abdominal, but increased scoliosis of the caudal vertebral column. Alterations were diagnosed based on Alizarin red S whole mount-stained specimens; only the abdominal and caudal region of the vertebral column were considered for the analysis. Chi-squared test followed by Bonferroni correction; p values are indicated; ns: non-significant. (C) Severity assessment of the maximal curvature index diagnosed in WT, Chi/+ and Chi/+_LP related to abdominal kyphosis and lordosis, and to caudal kyphosis, lordosis and scoliosis. The graph shows individual data points and the mean value (black bar) for the maximal curvature index. The maximal kyphotic and lordotic indices were calculated in the sagittal plane of Alizarin red S stained specimens as the ratio (a/b) between the perpendicular distance from the axis (in correspondence of the maximal curvature, segment ‘a’ in A) and the standard length (segment ‘b’ in A). The same method but in the coronal plane was used to calculate the maximal scoliotic index. Mann-Whitney test was applied with a minimum of three values per group; ns, non-significant.

Vertebral body shape variation in Chi/+ and Chi/+_LP mutants. (A) Alizarin red S stained vertebral body of a WT animal with 2D landmark positions used for quantifying the shape variation by means of geometric morphometrics, represented in (B). (B) The scatterplot of WT, Chi/+ and Chi/+_LP 2D landmarks shows high variation in the superimposition of X,Y Procrustes coordinates of Chi/+ compared to WT animals. Chi/+_LP animals display reduced landmark variation compared to Chi/+ and a distribution more similar to WT indicating a partial rescue of shape variation at three months of age. The first 10 caudal vertebral centra in WT (n=15), Chi/+ (n=13) and Chi/+LP (n=12) were analysed. The 95% confidence ellipses are shown. (C) Principal component analysis of vertebral centra shapes. Each symbol in the plot represents a vertebral body. PC indicates Principal Component and the values in the axis labels indicate the percentage of variation accounted for by each axis. Chi/+ animals show high variance compared to WT animals (Chi/+ versus WT, PC1 0.3321, PC2 0.1795; Chi-square test: p < 0.001). Variance is rescued in Chi/+_LP animals (Chi/+_LP versus WT, PC1 0.2484, PC2 0.2020; Chi-square test: non-significant). The 95% confidence ellipses are shown.

Histology of vertebral column of WT, Chi/+ and Chi/+_LP confirms the irregular shape of mutant vertebral bodies. (A) Schematic representation of the medio-sagittal plane of a zebrafish vertebral body centrum and two intervertebral spaces. Vertebral centra derive from segmental mineralisation of the notochord sheath and intramembranous bone formation around the notochord. Vertebral body endplates (en) are connected by intervertebral ligaments. Ligaments consist of the enlarged notochord sheath (ns, a collagen type II layer secreted by the cells of the notochord epithelium, ne), its outer elastin layer (el) and dense collagen type I fibre bundles (dc) produced by fibroblasts (fb) that surround the notochord. The collagen type I fibre bundles (cf) continue in the bone of the vertebral body endplates (en) as Sharpey fibres. Osteoblasts (ob) deposit new bone matrix that expands the vertebral body endplates in the bone growth zone. Inside, the notochord is composed of vacuolated notochord cells (nc) and extracellular vacuoles (ev). Condensed notochord cells constitute the notochord septum (se) and the notochord strand (st). Boxes indicate locations where the bone thickness was measured, i.e. endplates (1), central region of vertebrae (2) and trabecular bone (3). (B) Representative three months old WT and mutant sagittal sections of the vertebral column stained with toluidine blue. Compared to WT, Chi/+ mutants (representative also for Chi/+_LP) have several vertebral centra with deformed endplates that are shifted against each other along the dorsal-ventral axis (black arrowheads). Chi/+ animals also suffer from vertebral body compression fractures (red asterisk), scoliosis, lordosis and kyphosis. Scoliosis can be appreciated from the absence of a straight sagittal midline plane as seen in the WT animal. (C) Higher magnification of vertebral body endplates in WT and Chi/+ (representative also for Chi/+_LP) animals. Toluidine blue staining shows deformed endplates of adjacent vertebral bodies (red arrowheads) in Chi/+ mutants, yet with unaltered ligaments and unaltered intervertebral space as in WT.

Figure 4 Chi/+ vertebral bone structures are thin and highly mineralised. The LP diet restores the osteoid. (A) Sagittal histological non-demineralised sections stained with Von Kossa/Van Gieson show that three months old Chi/+ animals, compared to WT animals, have highly mineralised endplates. No osteoid layer can be identified. The osteoid (pink, black arrowheads) is restored in Chi/+_LP. Mineralised bone: black; dense collagen and non-mineralised bone: red. (B) Quantitative analysis of vertebral body endplate mineralisation (scored as low, intermediate or high) based on whole mount-stained specimens shows that Chi/+ animals exhibit a higher degree of mineralisation compared to WT animals. The LP diet reduces mineralisation of the vertebral body endplates in some Chi/+ individuals. The first 5 caudal vertebral centra in WT (n=15), Chi/+ (n=13) and Chi/+_LP (n=12) were analysed. Chi-square test followed by Bonferroni correction, p values are indicated, ns: non-significant. (C) Measurements of bone structure thickness at three locations: (i) vertebral endplates, (ii) central region of vertebrae and (iii) trabecular bone (see Figure 3A for locations). Compared to WT animals, Chi/+ and Chi/+_LP animals have thinner bone structures in all three locations (see also Table 2). Thickness of bone structures was measured on toluidine blue stained sections in 5 to 10 vertebral centra in WT (n=4), Chi/+ (n=4) and Chi/+_LP (n=5). Mann-Whitney test followed by Bonferroni correction, p values are indicated, ns: non-significant.

Different grades of Chi/+ and Chi/+_LP compression fractures. Whole mount Alizarin red S stained vertebral bodies of WT (A) and mutants (B–D) visualised in bright field (left) and with fluorescence (right). Inter-individual variability and different severity levels of compression fractures are observed in three months old mutant zebrafish. Bone calli associated to fractures appear more dense than other bone elements when visualised with fluorescent light. (B) Example of a mutant Chi/+_LP showing a compression fracture affecting only one vertebral body, bone callus is visible (white arrow). (C)Chi/+_LP zebrafish displaying kyphosis associated with multiple compression fractures and evident bone calli (white arrowheads). (D) Mutant Chi/+ fish displaying severely distorted vertebrae (black arrowheads) and a collapsed vertebral body (white asterisk). The inserts in (C, D) demonstrate the identification of osteoid on whole mount-stained specimens. Black arrows indicate the presence (C) and absence (D) of osteoid in Chi/+_LP and Chi/+, respectively.

Compression fractures and bone resorption in Chi/+ and Chi/+_LP vertebral bodies. (A) Toluidine blue stained medio-sagittal section of a compression fracture from three months old mutant zebrafish (representative for both Chi/+ and Chi/+LP) observed with polarised light. The compression fracture is characterised by several fractures in the central region of the vertebral body (black arrowheads); a bone callus is present on the outside of the vertebral centrum (white arrowheads). The fracture also disrupts the notochord tissue and induces condensation of chordocytes into a fibrous tissue (a known reaction of notochord tissue to injuries) (red asterisks). (B) High magnification of the fractured bone (black arrowheads) inside the notochord. The reaction of the notochord tissue can be seen (red asterisks). (C) A fibrocartilaginous callus is present around the fractured central part of the vertebral body, the typical appearance for fracture callus at initial stages of repair (white asterisks). Polarised light (insert) shows collagen fibres (green) within the cartilaginous callus. Red asterisk indicates the notochord tissue condensation. (D) Sagittal section of a healed compression fracture. Fracture repair and remodelling processes replaced the fibrocartilaginous tissue by a bone callus (white arrowhead). Remnants of the fibrocartilaginous tissue are visible (black arrow). (E) Tartrate-resistant acid phosphatase (TRAP) staining confirms compression fracture repair. TRAP activity (red staining, red arrowhead) indicates resorption of the fibrous tissue (white asterisks) that is being replaced by a hard bone callus. The fractured bone fragments (black arrowheads) in the lumen of the notochord do not show resorption, which is consistent with the absence of blood vessels, lymphatic vessels and innervation inside the notochord. (F) TRAP activity is detected also in the trabecular bone (white arrowheads) and vertebral endplate (white arrow) of a vertebra showing a compression fracture in mutant zebrafish (representative for both Chi/+ and Chi/+_LP). White asterisk indicates the fibrocartilaginous callus. (G, H) WT display TRAP activity (red) at sites of bone remodelling linked to bone growth, i.e. the endosteal surfaces of the neural (black arrowheads) and haemal arches (black arrow). NT, neural tube. (I, J)Chi/+ animals (representative also for Chi/+_LP) show TRAP activity at the same locations as in WT, however mutants exhibit expanded TRAP activity at all endosteal and periosteal bone surfaces, i.e. arches (black arrowheads) and bone trabeculae connecting the endplates (white arrowheads). NT, neural tube.

Ultrastructure of bone cells and bone matrix in Chi/+ and Chi/+_LP animals. (A) Transmission electron microscopy (TEM) of osteoclasts (OC) located at the arch surface of three months old WT and Chi/+ (representative for both Chi/+ and Chi/+_LP) vertebrae. The panel WT shows a typical flat-shaped teleost osteoclast (OC) characterised by its electrodense cytoplasm. The cell resides in a shallow resorption lacuna (dashed line), yet is attached to the bone surface (white arrow). The panel Chi/+ shows an osteoclast that resorbs the bone matrix and exhibits the typical ‘ruffled border’ (RB), an electron-dense cytoplasm with abundant cytoplasmic vacuoles and resorption vesicles (right panel, white arrowheads) in proximity of the ruffled border. N, nucleus. (B) TEM of WT, Chi/+ and Chi/+_LP shows mutant osteoblasts with enlarged endoplasmic reticulum (ER) cisternae (asterisks). The osteoblasts are located in the growth zone of the vertebral body endplate (see Figure 3A for location). Higher magnification images in (C) show that Chi/+ osteoblast ER cisternae are filled with protein, likely mutated collagen type I WT osteoblasts have numerous, yet not enlarged ER cisternae (arrows). ECM: extracellular matrix of the bone surface; N: nucleus. (C) The newly secreted collagen fibrils (white arrowheads) in the proximity of the osteoblasts in WT are visibly separated prior to maturation and assemblage into collagen fibres. In contrast, the Chi/+ animal has densely packed collagen fibrils. No space can be recognised between the fibrils. In the Chi/+_LP individual collagen fibres are densely packed and space between the fibrils is distinguishable in proximity to the osteoblasts. (D) Longitudinal sections of collagen fibres show a regular D-periodicity pattern in a WT animal, absence D-period pattern in a Chi/+ mutant, and a less regular D-periodicity pattern a Chi/+_LP specimen.