Collagen XI regulates the acquisition of collagen fibril structure, organization and functional properties in tendon

Abstract

Collagen XI is a fibril-forming collagen that regulates collagen fibrillogenesis. Collagen XI is normally associated with collagen II-containing tissues such as cartilage, but it also is expressed broadly during development in collagen I-containing tissues, including tendons. The goals of this study are to define the roles of collagen XI in regulation of tendon fibrillar structure and the relationship to function. A conditional Col11a1-null mouse model was created to permit the spatial and temporal manipulation of Col11a1 expression. We hypothesize that collagen XI functions to regulate fibril assembly, organization and, therefore, tendon function. Previous work using cho mice with ablated Col11a1 alleles supported roles for collagen XI in tendon fibril assembly. Homozygous cho/cho mice have a perinatal lethal phenotype that limited the studies. To circumvent this, a conditional Col11a1^flox/flox mouse model was created where exon 3 was flanked with loxP sites. Breeding with Scleraxis-Cre (Scx-Cre) mice yielded a tendon-specific Col11a1-null mouse line, Col11a1^Δten/Δten. Col11a1^flox/flox mice had no phenotype compared to wild type C57BL/6 mice and other control mice, e.g., Col11a1^flox/flox and Scx-Cre. Col11a1^flox/flox mice expressed Col11a1 mRNA at levels comparable to wild type and Scx-Cre mice. In contrast, in Col11a1^Δten/Δten mice, Col11a1 mRNA expression decreased to baseline in flexor digitorum longus tendons (FDL). Collagen XI protein expression was absent in Col11a1^Δten/Δten FDLs, and at ~50% in Col11a1^+/Δten compared to controls. Phenotypically, Col11a1^Δten/Δten mice had significantly decreased body weights (p < 0.001), grip strengths (p < 0.001), and with age developed gait impairment becoming hypomobile. In the absence of Col11a1, the tendon collagen fibrillar matrix was abnormal when analyzed using transmission electron microscopy. Reducing Col11a1 and, therefore collagen XI content, resulted in abnormal fibril structure, loss of normal fibril diameter control with a significant shift to small diameters and disrupted parallel alignment of fibrils. These alterations in matrix structure were observed in developing (day 4), maturing (day 30) and mature (day 60) mice. Altering the time of knockdown using inducible I-Col11a1^-/- mice indicated that the primary regulatory foci for collagen XI was in development. In mature Col11a1^Δten/Δten FDLs a significant decrease in the biomechanical properties was observed. The decrease in maximum stress and modulus suggest that fundamental differences in the material properties in the absence of Col11a1 expression underlie the mechanical deficiencies. These data demonstrate an essential role for collagen XI in regulation of tendon fibril assembly and organization occurring primarily during development.

Keywords: Col11a1; Collagen XI; Collagen fibrillogenesis; Conditional mouse model; Tendon; Tendon biomechanics; Tendon structure.

Fig. 1.

Strategy for creating tendon targeted Col11a1 conditional knockout mice. (A) Schematic diagram showing the wild-type allele, targeting vector, targeted allele, and floxed allele as well as the excised allele after Cre recombination of Col11a1 gene. The Col11a1 targeting vector was obtained from the KOMP Repository (project ID: CSD80258). Exon 3 of Col11a1 is flanked with LoxP sites and the Neo cassette is flanked with FRT sites. Location of primers for LacZ, as well as 3’ and 5’ primers used to determine insert orientation are shown (red arrows). The large blue and green arrows indicate the location of the primers used to identify specific alleles. (B) Strategy for gene targeting of embryonic stem cells and generation of tendon-targeted Col11a1 conditional-knockout mice. Linearized Col11a1 targeting vector was electroporeated into ES cells. Blastocysts were injected with targeted ES cells yielding chimeric mice. The chimeric mice were crossed with wild type mice yielding F1 progeny with the targeted allele (Col11a1^+/ta). Breeding of Col11a1^+/ta with FLPe mice resulted in excision of FRT flanked sequences, removal of the Neo cassette and creation of conditional knockout mice (Col11a1^flox/flox). These mice were bred with mice expressing ScxCre to target the knockout to tendons (Col11a1^Δten/Δten).

Fig. 2.

Analysis of tendon targeted Col11a1 knockout mice. (A) Characterization of the targeted Col11a1 ES cells using short-range PCR (left panel) and long-range PCR (right panel). Clone 1F11 is the targeted ES cell clone injected into blastocysts. (B) Genotyping of the targeted allele, and floxed alleles in different stages of creating the conditional Col11a1 knock-out mice. (C) Genotyping of the Scleraxis-Cre mice (Scx-Cre), heterozygous (Col11a1^+/Δten) and homozygous (Col11a1^Δten/Δten) tendon targeted Col11a1 knockout mice.

Fig. 3.

Knockout of Col11a1 mRNA and protein expression in the Col11a1 tendon targeted conditional mice. (A) Quantitative real-time PCR shows comparable expression of Col11a1 mRNA in the wild type and Col11a1^flox/flox controls. Col11a1 mRNA expression in Col11a1^Δten/Δten FDLs is reduced to baseline while expression in the Col11a1^+/Δten FDLs was ~50% of control values. Day 14 wild-type (n=3), Col11a1^flox/flox (n=5), Col11a1^+/Δten (n=8), Col11a1^flox/flox (n=7) mice for each genotype. (B) Western blot analysis of Col11a1 protein (α1(XI)) content in control; wild type (n=3), Scx-Cre (n=4), Col11a1^flox/flox (n=7) mice was done using Wes automated western blotting. The Col11a1^+/Δten (n=3), and Col11a1^Δten/Δten (n=4) FDL contained reduced and virtually no α1(XI) reactivity relative to controls. All 3 control mice contained comparable amounts of α1(XI). Day 4 mice. Left panel shows a representative image and the right panel presents the quantitation results from all mice.

Fig. 4.

Abnormal fibril structure in FDLs from tendon targeted Col11a1 knockout mice. (A-C) Transmission electron microscopy demonstrates a disruption in the parallel fibril alignment in FDLs in Col11a1^Δten/Δten mice compared to wild type mice while fibril alignment in Col11a1^+/Δten FDLs is partially disrupted. (D-F) Col11a1^Δten/Δten FDLs have a larger percentage of smaller, more heterogeneous and structurally abnormal fibrils compared with wild types controls. In contrast, haploinsufficient Col11a1^+/Δten FDLs show only a mild disruption of fibril structure. (G-I) All 3 distributions are significantly different from one another (p<0.001 K-S test). There is a striking difference between the Col11a1^Δten/Δten and the other 2 distributions. The fibril diameter distribution is significantly shifted to the smaller diameter fibrils with the larger diameter subpopulation reduced to a minor shoulder in the Col11a1^Δten/Δten mice compared to the wild type mice. The wild type and Col11a1^+/Δten distributions are very similar with the reduction in Col11a1 associated with a mild phenotype with an increase in large diameter fibrils relative to the control. Day 30 males, n=2 for each genotype.

Fig. 5.

Tendon targeted Col11a1 knockout mice are weaker. Fore-limb grip strength is significantly decreased in Col11a1^Δten/Δten mice (n=9) compared to Col11a1^flox/flox control mice (n=8). Day 60 male mice.

Fig. 6.

Altered biomechanical properties in FDLs in tendon targeted Col11a1 knockout mice. FDL tendons from tendon-targeted Col11a1^Δten/Δten null mice demonstrated significant alterations in the biomechanical properties. The Col11a1^Δten/Δten FDLs were smaller, weaker, and stiffer compared to the wild type controls. (A) A significant decrease in cross-sectional area was observed. In addition, (B) maximum load, (C) maximum stress, (D) stiffness, and (E) modulus were significantly reduced in the Col11a1^Δten/Δten mice compared to controls. Decreases in maximum stress and modulus suggest that fundamental differences in the material properties of Col11a1^Δten/Δten and control FDL tendons underlie the mechanical deficiencies. Day 60 male Col11a1^flox/flox (n=12) and Col11a1^{Δten/ Δten} (n=15) male mice.

Fig. 7.

The absence of Col11a1 expression results in abnormal FDL fibrils in mature Col11a1^Δten/Δten mice. FDLs from mature male Col11a1^Δten/Δten mice (day 60) have a severe disruption in collagen fibril structure and diameter distributions compared to control tendons. (A,B) Electron microscopic analysis demonstrates a substantial increase in small diameter fibrils as well as overall smaller diameters in the FDLs from Col11a1^Δten/Δten compared to control FDLs. (C-E) The fibril distributions from wild type and Col11a1^Δten/Δten tendons are significantly different (KS p<0.0001). In the Col11a1^Δten/Δten mice there is a significant shift to small diameter fibrils and a virtual absence of large diameter fibrils characteristic of the mature FDL. The mature day 60 mice show the same trends as seen in maturing day 30 mice (Fig. 4). (F) Fibril density is significantly increased in the Col11a1^Δten/Δten FDLs compared to controls, consistent with the significant shift to small diameter fibrils observed in this genotype. (n=3 mice for each genotype).

Fig. 8.

Expression of Col11a1 regulates early steps of fibrillogenesis. (A,B) Electron microscopic analysis demonstrates smaller and more heterogeneous fibrils in the FDLs from developing Col11a1^Δten/Δten mice compared with the wild type mice at day 4. The large diameter fibrils seen in Col11a1^Δten/Δten showed evidence of fusion with small diameter fibrils (arrows) suggesting their formation was a result of unregulated lateral fibril growth. (C-E) The diameter distributions from wild type and Col11a1^Δten/Δten FDLs are significantly different (KS p<0.0001). The fibril diameter distributions shows a substantial increase in small diameter fibrils and the broader distribution of fibril diameters in the Col11a1^Δten/Δten FDLs compared to controls. (F) Fibril density in Col11a1^Δten/Δten FDLs is similar to the controls, but considerably more variable consistent with the presence of a distinct subpopulation of very large diameter fibrils not seen in the control tendons. (n=3 mice for each genotype).

Fig. 9.

Induced knockdown Col11a1 in the mature tendon does not affect fibril structure. Knockdown of Col11a1 was induced using tamoxifen once daily for 3 days (i.p 10mg/100g body weight) beginning at day 25 and fibril structure analyzed at day 60. This approach allows the FDLs to develop as in wild type through development and into maturation. Knockdown is during maturation and mature FDLs are analyzed. (A,B) Electron microscopic analysis shows no significant effect on the fibril diameter and organization. Fibrils from both genotype have normal circular profiles. (C-E) The diameter distributions from wild type and I-Col11a1^−/− FDLs are both bimodal and very similar. The larger diameter subpopulation is shifted modestly toward larger diameter in the I-Col11a1^−/− compared to wild type FDLs. (F) Fibril density in I-Col11a1^−/− FDLs is decreased, consistent with the increase in diameter observed. (n=3 mice for each genotype).