Targeted conditional collagen XII deletion alters tendon function

Abstract

Collagen XII is a fibril-associated collagen with interrupted triple helices (FACIT). This non-fibrillar collagen is a homotrimer composed of three α1(XII) chains assembled into a collagenous molecule with a C terminal collagenous domain and a large N terminal non-collagenous domain. During tendon development and growth, collagen XII is broadly expressed throughout the extracellular matrix and enriched pericellularly around tenocytes. Tendons in a global Col12a1 ^-/- knockout model demonstrated disrupted fibril and fiber structure and disordered tenocyte organization, highlighting the critical regulatory roles of collagen XII in determining tendon structure and function. However, muscle and bone also are affected in the collagen XII knockout model. Therefore, secondary effects on tendon due to involvement of bone and muscle may occur in the global knockout. The global knockout does not allow the definition of intrinsic mechanisms involving collagen XII in tendon versus extrinsic roles involving muscle and bone. To address this limitation, we created and characterized a conditional Col12a1-null mouse model to permit the spatial and temporal manipulation of Col12a1 expression. Collagen XII knockout was targeted to tendons by breeding conditional Col12a1 ^flox/flox mice with Scleraxis-Cre (Scx-Cre) mice to yield a tendon-specific Col12a1-null mouse line, Col12a1 ^Δten/Δten . Both mRNA and protein expression in Col12a1 ^Δten/Δten mice decreased to near baseline levels in flexor digitorum longus tendons (FDL). Collagen XII immuno-localization revealed an absence of reactivity in the tendon proper, but there was reactivity in the cells of the surrounding peritenon. This supports a targeted knockout in tenocytes while peritenon cells from a non-tendon lineage were not targeted and retained collagen XII expression. The tendon-targeted, Col12a1 ^Δten/Δten mice had significantly reduced forelimb grip strength, altered gait and a significant decrease in biomechanical properties. While the observed decrease in tendon modulus suggests that differences in tendon material properties in the absence of Col12a1 expression underlie the functional deficiencies. Together, these findings suggest an intrinsic role for collagen XII critical for development of a functional tendon.

Keywords: Biomechanics; Col12a1; Collagen XII; Conditional mouse model; Tendon; Transgenic.

Fig. 1

Strategy for creation of a tendon targeted Col12a1 conditional knockout mouse model. A targeted Col12a1 ES cell line, Col12a1^{tm2a(KOMP)Wtsi}, was obtained from the KOMP Repository (project ID: CSD29388). (A) This schematic diagram illustrates the wild-type Col12a1 allele; the targeted allele where exons 15–16 are flanked with LoxP sites and the Neo cassette is flanked with FRT sites; the floxed allele after removal of the FRT flanked Neo cassette; and the excised allele after Cre recombination of the Col12a1 gene. Location of primers for LacZ, as well as 3′ and 5′ primers used to determine insert orientation are shown (red arrows). (B) Genotyping of the targeted allele and floxed alleles in different stages of creating the conditional Col12a1 knock-out mice. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 2

Genotyping analysis of tendon targeted Col12a1 knockout mice. Genotyping of the control mice; wild type (WT), Scleraxis-Cre (Scx-Cre), floxed (Col12a1^f/f) as well as targeted heterozygous (Col12a1^+/Δten) and homozygous (Col12a1^Δten/Δten) mice was used to characterize the targeted models.

Fig. 3

Knockout of Col12a1 mRNA expression in tendon targeted conditional mice. Quantitative real-time PCR analyses of Col12a1 mRNA expression in Col12a1^Δten/Δten FDLs is reduced to baseline while expression in the Col12a1^+/Δten FDLs was ∼ 25% of control values. Day 10 Control (n = 8, including WT n = 4, Scx-Cre n = 2, Col12a1^+/flox n = 2), Col12a1^+/Δten (n = 3), Col12a1^Δten/Δten (n = 3), and Col12a1^-/- (n = 1) mice (*p < 0.05).

Fig. 4

Knockout of collagen XII expression in the Col12a1 tendon targeted conditional mice. Analysis of Col12a1 protein (α1(XII)) content in ; Control (n = 5, including WT n = 3, Scx-Cre n = 1, Col12a1^+/flox n = 1), Col12a1^+/Δten (n = 3), Col12a1^Δten/Δten (n = 3) mice, as well as global knock out mice, Col12a1^-/- (n = 1) was done using Wes automated western blotting. The Col12a1^+/Δten and Col12a1^Δten/Δten FDLs in day 10 mice contained reduced and virtually no α1(XII) reactivity relative to controls. (A) shows a representative image and (B) presents the quantitation of results from all mice (**p < 0.01, *p < 0.05).

Fig. 5

Collagen XII in wild type and Col12a1^Δten/Δten FDLs. Immuno-localization analyses of collagen XII demonstrated an absence of reactivity in the tendon proper of Col12a1^Δten/Δten compared to wild type FDLs. In contrast, cells of the surrounding peritenon (white arrows) show reactivity for collagen XII in both genotypes. Day 14 Control (Col12a1^flox/flox, n = 2) and Col12a1^Δten/Δten (n = 2) FDLs.

Fig. 6

Smaller size and altered gait in Col12a1^Δten/Δten mice. (A) Representative gross images showing that Col12a1^Δten/Δten mice are smaller than control Col12a1^+/+ and heterozygous Col12a1^+/Δten mice, with no obvious body size difference between control and heterozygous mice. All mice are male litter mates at 60 days. (B) Representative gait analysis showed shorter stride length in Col12a1^Δten/Δten mice compared to wild type controls. However, all mice walk with regular, even steps, and there is no obvious change in the width of toe spread. The arrowed vertical lines indicate the stride lengths from the 3rd toe to adjacent 3rd toe of the same hind foot. Male 60 day mice. (C) Quantitative presentation of the stride length data from (B). There was a significant decrease in stride length in Col12a1^Δten/Δten mice compared to heterozygous and wild type mice. There was no difference in the latter two groups. (*p < 0.05).

Fig. 7

Tendon-targeted Col12a1 knockout mice are weaker. Fore-limb grip strength is significantly decreased in Col12a1^Δten/Δten male mice (n = 8) compared to Col12a1^flox/flox control mice (n = 5). Mature male and female mice were tested at day 65 to 90, n = 5–8 mice per genotype (**p < 0.01).

Fig. 8

Altered biomechanical properties in FDLs in tendon targeted Col12a1 knockout mice. FDL tendons from tendon-targeted Col12a1^Δten/Δten null mice demonstrated significant alterations in the biomechanical properties. No differences in (A) cross-sectional area were observed between genotypes. However, (B) stiffness and (C) modulus were significantly reduced in Col12a1^Δten/Δten mice compared to controls, while there were no differences in (D) stress relaxation at 5% strain. Decreases in modulus suggest that fundamental differences in the material properties of Col12a1^Δten/Δten and control FDL tendons underlie the mechanical deficiencies. Mature day 60 male Col12a1^flox/flox (n = 7), Col12a1^+/Δten (n = 9) and Col12a1^Δten/Δten (n = 9), and female Col12a1^flox/flox (n = 4), Col12a1^+/Δten (n = 8), and Col12a1^Δten/Δten (n = 6) mice were tested (**p < 0.01, *p < 0.05).