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. Author manuscript; available in PMC: 2022 Aug 9.
Published in final edited form as: Nat Cell Biol. 2020 Jan 6;22(1):74–86. doi: 10.1038/s41556-019-0441-z

Figure 1. Circadian regulation of the tendon collagen extracellular matrix.

Figure 1

(A) Representative transverse TEM images of Achilles tendon at ZT3 (n=1134 fibrils) and ZT15 (n=2262 fibrils) (n=6 mice.) Bars 200 nm. (B) Transverse section of Achilles tendon showing characteristic D1, D2 and D3 fibrils. (C) Shift in the trimodal distribution of fibril diameters at ZT3 and ZT15. (D) Changing fibril volume fractions of D1, D2 and D3 fibrils during 24 hours. (E) STEM mass mapping electron microscopy data from mechanically dispersed entire Achilles tendons (n=4 mice and 1000 D1 fibrils measured at each time point) showing the variation of the D1 fibril component at 3-hour time intervals during 24 hours. This is in the form of a double plot with the data repeated over 48 hours. The data were fitted by least squares minimization to a sinusoidal variation (red line). The grey panels indicate the night-time (dark) periods. (F) Still image from a step-through movie generated by serial block face-scanning electron microscopy of 6-week old mouse Achilles tendon harvested at CT3 (Supplementary Supplementary Video 1). Selected from n=12 reconstructions from 12 different mice. (G) Still from a step-through 3D reconstruction displaying individual fibrils in a small region of mouse tail tendon, showing merging of D1 fibrils onto larger D2 and D3 fibrils (Supplementary Video 2). Fibrils that appear in at least 80% of frames are coloured. Images generated from serial block face-scanning electron microscope stack of transverse TEM images. Area shown is 4 μm by 4 μm, each frame is a longitudinal step of 100 nm through the tendon. (H) Selected images from a serial block face-scanning electron microscope step-through movie stack with a D3 fibril (outlined) surrounded by a D2 (dark blue) fibril and nine D1 fibrils (shown in different colours). Cut numbers are shown. (I) Circularity of collagen fibrils examined in transverse TEM images of Achilles tendons from ZT3 and ZT15 indicate fibril cross sections from ZT3 are more irregular than fibril cross sections from ZT15 (n=1000 fibrils measured in both ZT3 and ZT15 samples ****p=2×10-48, two-tailed unpaired t-test. Bars = mean ± SEM. See also Source Data for Figure 1.