Figure 2

Figure 2

Mineralisation levels of vertebral column after two months of dietary treatment. (a) Vertebral column of low P diet (LP) treated animals is characterised by reduced radiodensity compared to controls (RP) and high P diet (HP) treated animals. HP zebrafish present multiple vertebral body fusions (white arrowheads). Scale bar: 1 mm. (b) Alizarin red S staining of vertebral columns shows vertebral body endplates (black arrowheads) characterised by low mineralisation levels in LP animals, intermediate mineralisation in controls and high mineralisation levels in HP animals. HP zebrafish present vertebral body fusion (white arrowhead). Neural (na) and haemal arches (ha) are low-mineralised and their spines (ns, hs, respectively) are deformed in LP individuals compared to RP and HP zebrafish. Scale bar: 200 µm. (c) Quantitative analysis of vertebral body endplate mineralisation: the non-mineralised endplate is expressed as percentage of the total non-mineralised endplate length (A + A’) over the total vertebral length (B), (A + A’)/B. Chi squared or Fisher’s exact test, pairwise comparison, *: p < 0.05. (d) Qualitative analysis of arch mineralisation levels. Please see Materials and Methods for further details. Chi squared or Fisher’s exact test, pairwise comparison, *: p < 0.05. (e) Sagittal sections of vertebral bodies show large areas of non-mineralised matrix at the level of the vertebral endplates (black arrows) in LP animals compared to RP and HP animals. LP individuals present also a thin osteoid layer in the outer part of the vertebral body (black arrowheads), completely absent in RP and HP animals. Scale bar: 100 µm. (e’) High magnification of endplates in panel e. Extended non-mineralised bone matrix (black arrows) is visible in the vertebral endplates of LP animals. RP and HP zebrafish present thin endplates with reduced osteoid (black arrows). White arrowheads indicate intervertebral ligaments (see Figure 6a for details). Scale bar: 20 µm. (f) Mineralisation of bone trabeculae (asterisks) is also affected by low P levels in the diet. Black arrows: endplates. Von Kossa/Van Gieson staining: mineralised bone: brown; non-mineralised collagen matrix/osteoid: red. Scale bar: 100 µm.