In-depth characterization of the ultrastructural variability of the vertebral column in a severe, medium and mildly affected Osteogenesis Imperfecta zebrafish model

Osteogenesis Imperfecta (OI) is mainly caused by dominant mutations in the COL1A1 and COL1A2 genes, which encode the pro-1 and pro-2 chains of type I (pro)collagen of bone, and which mostly result in glycine substitutions. The disease complex is characterized by an enormous clinical variability ranging from mild forms with few fractures to lethal forms with severe deformities of the skeleton. In recent years, zebrafish has gained popularity as OI model to study OI in humans. Importantly, zebrafish have heterogenous genomes expressing a similar variability in OI bone phenotypes compared to humans. To dissect the ultrastructural basis of phenotypic variability in different forms of dominant OI, the intervertebral ligament was investigated in three previously characterized OI zebrafish models with different phenotypic severity: mh15 (severe phenotype), dc124 (medium phenotype) and mh13d (mild phenotype). These OI models contain different glycine substitutions in the type I collagen alpha chains. First the intervertebral ligament of wild type (WT) zebrafish was further characterized on parasagittal transmission electron microscopy (TEM) sections. Second, to investigate the level of malformation of the intervertebral ligaments in mutants, morphological parameter scores were compared between mutants and their matched WT siblings. Finally, the severe, medium and mild mutant zebrafish models were compared. The characterization of the intervertebral ligament in WT zebrafish allowed three groups of parameters to be defined: (i) number of cells, (ii) thickness and number of collagen layers, (iii) grading of key structures. Group one parameters were the number of chordoblasts, the total number of cells in the intervertebral ligament, cells on the mature collagen and cells on the bone of the vertebral endplate. Group two parameters were thickness of the mature collagen layer, number of alternating layers in the mature collagen layer, thickness of longitudinal collagen layers, thickness of circumferential collagen layers and bone thickness. Group three parameter were intervertebral ligament left-right angle deviation, elastin grade, endoplasmic reticulum grade and autocentrum grade. A significantly higher number of cells in the intervertebral ligament and on the mature collagen were observed in mh13 mutants. A significantly higher autocentrum grade was observed in mh15 mutants, while thinner bone was observed in all OI mutants. Mh15 mutants had indeed most severely deformed intervertebral ligaments, followed by dc124 and Mh13 mutants, respectively. In conclusion, these results argue for zebrafish as a promising tool in future research aiming to dissect the underlying basis of phenotypical variability in human skeletal disorders, such as OI.