Col11a1 Regulates Bone Microarchitecture during Embryonic Development

Abstract

Collagen XI alpha 1 (Col11a1) is an extracellular matrix molecule required for embryonic development with a role in both nucleating the formation of fibrils and regulating the diameter of heterotypic fibrils during collagen fibrillar assembly. Although found in many different tissues throughout the vertebrate body, Col11a1 plays an essential role in endochondral ossification. To further understand the function of Col11a1 in the process of bone formation, we compared skeletal mineralization in wild-type (WT) mice and Col11a1-deficient mice using X-ray microtomography (micro-CT) and histology. Changes in trabecular bone microstructure were observed and are presented here. Additionally, changes to the periosteal bone collar of developing long bones were observed and resulted in an increase in thickness in the case of Col11a1-deficient mice compared to WT littermates. Vertebral bodies were incompletely formed in the absence of Col11a1. The data demonstrate that Col11a1 depletion results in alteration to newly-formed bone and is consistent with a role for Col11a1 in mineralization. These findings indicate that expression of Col11a1 in the growth plate and perichondrium is essential for trabecular bone and bone collar formation during endochondral ossification. The observed changes to mineralized tissues further define the function of Col11a1.

Keywords: Col11a1; Collagen XI; X-ray microtomography; chondrodysplasia; mineralization.

Figure 1

Micro-CT images of whole body for WT and Col11a1-deficient littermates at embryonic day 17.5 (e17.5d). (A) WT mouse; and (B) Col11a1-deficient mouse. Differences between WT and the Col11a1-deficient mice were consistent with previous characterization, which focused on changes to the cartilage. These images are representative of all WT and mutant mice included in this study.

Figure 2

Three-dimensional reconstructions from X-ray micro-CT data of axial skeleton and ribs. (A and C) WT mouse; and (B and D) Col11a1-deficient mouse; Differences in spinal curvature and length are apparent upon comparison, as well as a decrease in the separation between vertebrae in the mutant mouse. Lumbar vertebrae from the Col11a1-deficient mouse were less mineralized than the WT mouse and were not visible by micro-CT. These images are representative of all WT and mutant mice included in this study. Scale bar A = 800 μm; scale bar B = 950 μm; scale bar C = 2500 μm; scale bar D = 2200 μm.

Figure 3

Pairwise comparison of shape and size of individual vertebrae. (A) Cervical vertebrae C2 through C7. (B) Thoracic vertebrae T1 through T7. (C) Thoracic vertebrae T8 through T13. Differences in shape and surface characteristics were apparent. Vertebral bodies of T8–T11 were less mineralized in the Col11a1-deficient mouse compared to WT. Vertebral bodies in WT mouse in comparison to Col11a1-deficient mouse show evidence of hemi-vertebrae malformation with decreased mineralization along the midline of the vertebra. Scale bar A–C = 400 μm.

Figure 4

Densitometric analysis of vertebral arch mineralization from the thoracic region. (A) Percent bone volume (BV/TV) was determined for each thoracic vertebral arch and values were averaged among six wild-type and three Col11a1^−/− e17.5d embryos. Col11a1^−/− (grey) vertebral arches had a greater percent bone volume consistently comparer to wild-type littermates (black). (B) Trabecular thickness (μm) was determined for each thoracic vertebral arch and values were averaged among six wild-type and three Col11a1^−/− e17.5d embryos. Trabecular thickness was consistently greater in the Col11a1^−/− embryos compared to wild-type littermates. (C) Trabecular number (1/mm) was determined for each thoracic vertebral arch and values were averaged among six wild-type and three Col11a1^−/− e17.5d embryos. No significant difference in trabecular number was observed between wild-type and Col11a1-deficient embryos. (D) Trabecular separation (μm) was determined for each thoracic vertebral arch and values were averaged among six wild-type and three Col11a1^−/− e17.5d embryos. No significant difference in trabecular number was observed between wild-type and Col11a1-deficient embryos. Error bars represent mean ± SD. Average values of all differences are presented in Table 1.

Figure 5

Densitometric analysis of the vertebral bodies from the thoracic region. (A) Percent bone volume (BV/TV) was determined for each thoracic vertebral body and values were averaged among six wild-type and three Col11a1^−/− e17.5d embryos. Col11a1^−/− (grey) vertebral bodies had a greater percent bone volume consistently comparer to wild-type littermates (black). (B) Trabecular thickness (μm) was determined for each thoracic vertebral body and values were averaged among six wild-type and three Col11a1^−/− e17.5d embryos. Trabecular thickness was consistently greater in the Col11a1^−/− embryos compared to wild-type littermates. (C) Trabecular number (1/mm) was determined for each thoracic vertebral body and values were averaged among six wild-type and three Col11a1^−/− e17.5d embryos. No significant difference in trabecular number was observed between wild-type and Col11a1-deficient embryos. (D) Trabecular separation (μm) was determined for each thoracic vertebral body and values were averaged among six wild-type and three Col11a1^−/− e17.5d embryos. While no significant difference in trabecular separation was observed between wild-type and Col11a1-deficient embryos for many of the individual vertebral bodies, there are some for which a significant decrease was detected. Error bars represent ± SD. Average values of all differences are presented in Table 2.

Figure 6

Comparison of ribs between WT and Col11a1-deficient mice. (A, C, E, G); WT (B, D, F, H); Col11a1-deficient mouse. (A and B); Histological differences in four adjacent ribs for WT and Col11a1-deficient mice. Trichrome staining rendered mineralized tissue green, cartilage tissue deep blue, and blood cells pink/purple. (C and D); Histological differences within proliferative/hypertrophic chondrocytes from WT and Col11a1^−/−. (E and F); Col11a1-deficiency led to an increase in mineralization in the newly-formed bone collar in the Col11a1-deficient mouse compared to WT. Images in C, D, E, F are from representative ribs not appearing in A and B. (G and H); Reduced length, increased curvature of the ribs was apparent in the Col11a1-deficient mice compared to WT. Proximal is oriented to the left for each rib, with the distal growth plate located on the right. Scale bars A and B = 0.5 mm; C and D = 0.1 mm; E and F = 1.0 mm.

Figure 7

Histological differences in the humeri of WT and Col11a1-deficient mice. (A, C, E, G); WT; (B, D, F, H); Col11a1-deficient mice. Trichrome staining was used to identify mineralized tissue (green), compared to cartilage tissue (blue). (A and B); Upper and lower hypertrophic and mineralized zone, with diaphysis and both growth plates shown for the Col11a1-deficient humerus. (C and D); Transition from hypertrophic to mineralized zone demonstrating altered hypertrophic cell size, a more abrupt transition from hypertrophic cartilage to mineralized tissue, and altered bone deposition. (E and F); Newly formed bone collar adjacent to the upper hypertrophic region demonstrates a thicker mineralized region adjacent to the cartilage in the mutant compared to WT. (G and H); Bone collar near the diaphysis from WT and Col11a1-deficient mice. Scale bars = 0.1 mm.

Figure 8

X-ray micro-CT images of forelimbs. (A and B); Col11a1-deficient mouse. (C and D); WT mouse; (A) Radius, ulna, and humerus of Col11a1-deficient mouse. The mineralized portions of the bones were shorter and wider in the Col11a1-deficient mice compared to (C) WT littermates (Table 3). Deltoid tuberosity was apparent in the WT humerus but absent in the Col11a1-deficient mouse. (B and D); Longitudinal cross-sections of each forelimb, shown in A and C, respectively. Mineralized tissue was assigned a color dependent upon three density ranges: low-density range (green), intermediate-density (blue), and high-density (white). Marrow space within the Col11a1-deficient limb showed regions of higher bone density near the proximal growth plates and those of very low density near the distal growth plates when compared to analogous regions in the WT littermate. (E and F); Cross-section of humeri at diaphysis, distal, and proximal metaphyses. (E) WT (F) Col11a1-deficient mouse. Col11a1-deficient humerus was wider and more cylindrical than WT. Mineralized tissue was assigned a color dependent upon three density ranges: low-density range (green), intermediate-density (blue), and high-density (white). Trabecular bone was denser in Col11a1-deficient mice compared to WT at proximal metaphysis. Trabecular bone is less dense in Col11a1-deficient mice compared to WT at distal metaphysis. Bone collar is less dense but thicker in Col11a1-deficient mice compared to WT littermate. Scale bars = 0.5 mm.