Unraveling of the ultrastructural features that determine the variability in phenotypic severity in different dominant forms of OI, using the zebrafish as a model

OBJECTIVES: Large phenotypic variability is observed between OI patients carrying different mutations in type I collagen. However, how specific structural mutations determine phenotypic severity is unknown. Therefore, we compared skeletal phenotypes of three OI zebrafish models, carrying different glycine substitutions in type I collagen (col1a1adc124/+, col1a2mh15/+, col1a1amh13/+) that show variability in phenotypic severity. The skeletal phenotype is studied employing a deep phenotyping workflow, by which multiple techniques can be applied on a single individual zebrafish so that the data can be integrated on specimen level, and by which an increasing level of detail is being harnessed with respect to disease severity and specific data on the skeleton. METHODS: For structural phenotyping whole-mount staining with Alizarin red S for mineralized tissues, scoring deformities, and the random forest model were used. Micro-CT, transmission electron (TEM), and quantitative backscatter (qBEI) analyses were used for ultrastructural phenotyping. RESULTS: Alizarin red S staining revealed most fractures occurring in col1a1adc124/+ and col1a2mh15/+ mutants with especially a high frequency of fusions occurring in the col1a2mh15/+ mutant, whereas the col1a1amh13/+ mutant showed a high frequency of compressions. Phenotypic scoring data of deformities was fed into a random forest model that could distinguish each zebrafish OI mutant model with intervertebral disc anomalies as one of the key diagnostic deformities. Micro-CT data indicated a slightly higher tissue mineral density (TMD) for the col1a1adc124/+ mutant, a significantly higher TMD in the col1a2mh15/+ mutant, and a lower TMD for the col1a1amh13/+ mutant. Bone analysed by qBEI of the col1a1adc124/+ mutant mainly showed higher Ca-peak values while a higher mineralization heterogeneity was noted in col1a2mh15/+ and col1a1amh13/+ mutants. Ultrastructural analysis showed disorganised collagen fibrils in the col1a1adc124/+ and col1a2mh15/+ mutants, with significantly thinner bone in the col1a1adc124/+ mutant. The col1a1amh13/+ mutant showed less disorganised collagen fibrils. CONCLUSION: We show that specific bone abnormalities result in different phenotypic severities in OI zebrafish models. The col1a1adc124/+ mutant, representing the most severe phenotype, shows severe skeletal deformities, disorganised thin bone matrix with high mineralisation. The col1a2mh15/+ mutant shows more variable skeletal deformities and disorganised but highly mineralised bone matrix, while the col1a1amh13/+ mutant, representing the mildest phenotype, reveals undermineralisation.