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.
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