Effects of col11a2-/- knockout in zebrafish.

The col11a2 -/- zebrafish have a fused vertebral column, ruptured annulus fibrosus and morphological abnormalities. (a) Micro-computed Tomography (μCT) of 6-month wild–type and mutant fish. The region highlighted with dashed yellow box is shown with higher magnification on the right panel. Note vertebrae fusions (magenta dash lines). (b) Fluorescent microscopic images of the calcified tissues of zebrafish labelled by Alizarin Red staining. Note abnormal vertebral trabeculations (arrow) and bone outgrowths from the arches (dashed arrow). Scale bars = 500 μm. (c) Toluidine blue stained images showing the cellular components of the soft tissue intervertebral disc (IVD) region. The top schematic (left) shows the encounter of two vertebrae at the IVD region, with the annulus fibrosus (AF) and nucleus pulposus (NP) annotated. The top right schematic shows a higher magnification view of the annulus fibrosus (AF) region, which is formed by an external layer of collagen fibres (co), an elastin layer (el) and another collagen rich region (co). The vertebral bone (b) and the notochord sheath (ns) are annotated. The sections of the wild–type and col11a2 -/- fish are shown in the middle and bottom rows respectively. Note that the elastin (el) and the collagen (co) layers are ruptured (arrow). (d) Forward and backwards Second Harmonic Generation (SHG) images of the wild–type and col11a2 -/- zebrafish intervertebral disc area. Note thicker collagen fibres at the endplates (dashed line) of mutant fish. Disorganised collagen fibres in the IVD (AF) is detected in mutants (arrowhead) Scale bars = 50μm.

Differential swimming shape analysis of col11a2 -/- zebrafish.

(a) illustration of our experimental setup for recording the 2d movement of fish. (b) Typical trajectories of mutant fish and wild–type fish replotted on top of the captured image. (c) Distribution of the bending index calculated from the image of the fish. The top panel, sharing the same x-axis with respect to the distribution plot, shows the characteristic shapes at different bending index values. (d) Average bending indices, calculated from the images of the fish shapes, for the wild–type fish and the mutant fish at different speed values. (e) Distribution of the speed values. Error bars represent the standard error calculated from 5 different fish.

The behaviour of wild–type fish and mutant fish swimming in a three dimensional space.

(a) A schematic of the experimental apparatus. The fish were placed in a bowl-shaped tank, and their movements were recorded by three synchronized cameras. The 3d trajectories of the fish were calculated from the recorded videos. The coloured line plots represent the movement of 25 mutant fish in 5 seconds. (b) The swimming trajectories for a single wild–type and a single mutant fish re–projected onto the recorded image, highlighting the difference in the persistence of the motion. (c, d) The joint probability distribution of the latitude radius (R) and the height (Z) of the wild–type fish and the mutant fish. (e, g) The probability distribution of the average swimming speed. (f, h) The averaged auto-correlation function of the fish orientation. (i) The probability distribution of the polarization order parameter (Eq 1), whose definition is illustrated on the left. The solid black line represents the polarization distribution of 25 randomly orientated vectors.

Important lengthscales of 25 wild–type fish (○) and mutant fish (▫).

(a) The nearest neighbour distance and the persistence length for the fish. Each scatter represents the average value in 2 minutes, whose grayscale values represent the age of the fish. (b) The relationship between the group polarisation and the reduced persistence length values. The data points represent the experiments and the line represents the simulation result where the fish obey the updating rule in Eq 2. The shading represents the regimes of the reduced persistence length which are dominated by the wild–type (red) and mutant (green). The dashed line represents the expected polarization of 25 randomly orientated vectors.