Fig. 1

Evaluation of skeletal anomalies in WT, col1a2^SNP/+, col1a2^VUS/+, and col1a2^mh15/+ zebrafish at 10 mpf. A Overview images of three representative zebrafish for each genotype stained with alizarin red. The white dotted line traces the curvature of the vertebral column, and the eye diameter is indicated by a white line for the col1a2^SNP/+ zebrafish. The white boxes highlights the region magnified in panel B. Scalebar = 5 mm. B A detailed view of the caudal vertebrae reveals no skeletal abnormalities in the col1a2^VUS/+ zebrafish while the positive control col1a2^mh15/+ shows vertebral fusions (f), compressions (c), fractures (arrow), as well as mineralization of the notochord (*) and IVL (arrowhead). Intramuscular bones are present across all genotypes (open arrow). In the WT group, they are consistently present but appear less distinct, likely due to being out of focus. Scalebar = 500 µm. C Skeletal phenotyping results. The X-axes represent the genotype. For eye diameter, the Y-axis represents the absolute diameter. For vertebral fusions and compressions, fractures, notochord mineralization, and IVL the Y axis represents the total number of occurrences of each deformity in the vertebral bodies per individual, excluding the first four vertebrae (forming the Weberian apparatus) and the last four vertebrae (preural and ural vertebrae). For scoliosis, zebrafish exhibiting scoliosis are shown in white, and those without scoliosis are shown in red. The number of zebrafish analyzed: WT=10, col1a2^SNP/+ = 13, col1a2^VUS/+ = 20, col1a2^mh15/+ = 11. Statistical analysis of scoliosis incidence was performed using the Fisher’s exact test (P < 0.001). All other parameters were analyzed by one-way ANOVA followed by Tukey’s multiple comparison test to WT. P-values of the Tukey’s multiple comparison tests to WT are the following for: Eye diameter (P < 0.0001), Fusion and compressions (P < 0.01), Fractures (P < 0.01), Notochord mineralization (P < 0.0001), IVL mineralization (P < 0.0001). Asterisks denote significance levels: **p<0.01, ***p<0.001, ****<0.0001. Data are presented as the mean ± standard deviation (SD)