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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 8. Circadian clock deficient tendons have abnormal collagen homeostasis.

Figure 8

(A) Luminometry recordings of endogenous circadian rhythms and re-initiation of the rhythms with dexamethasone (arrow) evident in dissected Achilles and tail tendons of wild type mice but not of Scx-Cre::Bmal1 lox/lox mice bred on a PER2::Luciferase background. A residual rhythm observed in the Scx-Cre::Bmal1 lox/lox tendons was likely due to cells that were not derived from Scleraxis-lineage cells. Relative amplitude and period of wild type and Scx-Cre::Bmal1 lox/lox tail and Achilles tendon circadian rhythms at 6-weeks of age (n=5 tail tendons, n=4 Achilles tendons; ***p=0.002, *p=0.0431, two-tailed unpaired t-test). Bars show SEM. (B) Representative transverse transmission electron microscopy images of tail tendons from 6 weeks-old wild type, ClockΔ19 and Scx-Cre::Bmal1 lox/lox mice showing irregular fibril cross sections in mutant tendons. Bars, 200 nm. (C) Fibril diameter distributions measured from transverse TEM images of wild type, ClockΔ19 and Scx-Cre::Bmal1 lox/lox tail tendons showing abnormal distribution of fibril diameters and increase in large (>400-nm fibrils) in mutant tendons (n=3 animals measured for each genotype; 1400 fibrils measured for each mouse; representatives for each genotype are shown). Red lines show 3-Gaussian fit curves. (D) Circularity of D1, D2 and D3 collagen fibrils of tail tendons sampled from ClockΔ19 and Scx-Cre::Bmal1 lox/lox mice compared to wild type littermate controls (images from n=3 animals measured for each genotype; total of 192 wild type, 179 ClockΔ19, 663 wildtype and 506 Scx-Cre::Bmal1 lox/lox fibres measured and shown. D1, D2 and D3 populations in mutants compared to same populations in littermate controls. Bars show SEM. (E) ClockΔ19 tendons are mechanically weaker (n=6 mice (means calculated from 10 tendons measured for each mouse); two-tailed unpaired t-test, *p=0.0328, *p=0.0170 ns: p=0.5333) and) and thicker (n=6 mice (means calculated from 10 tendons measured for each mouse); **p=0.0292) than age-matched (6-weeks old) wild type mice. Fibril volume fraction is unchanged in ClockΔ19 tendons compared to wild type tendons. Bars show SEM. (F) Scx-Cre:Bmal1 lox/lox tendons are mechanically weaker (n=6 mice (means calculated from >10 tendons measured for each mouse); two-tailed unpaired t-test, **p=0.0050, ***p=0.0004, **p=0.0073, nsp=0.3317) and thicker (n=6 mice (means calculated from 10 tendons measured for each mouse); **p=0.0050) than age-matched (6-weeks old) wild type mice. Bars show SEM. See also Source Data for Figure 8.