Cartilage abnormalities associated with defects of chondrocytic primary cilia in Bardet-Biedl syndrome mutant mice

Abstract

Primary cilia are found on nearly every mammalian cell, including osteocytes, fibroblasts, and chondrocytes. However, the functions of primary cilia have not been extensively studied in these cells, particularly chondrocytes. Interestingly, defects in the primary cilium result in skeletal defects such as polydactyly in Bardet-Biedl syndrome (BBS), a ciliary disorder that also results in obesity, retinopathy, and cognitive impairments. Wild-type mice and mutant mice of the ciliary proteins Bbs1, Bbs2, and Bbs6 were evaluated with respect to histological and biochemical differences in chondrocytes from articular cartilage and xiphoid processes. Using immunofluorescence microscopy, chondrocytic cilia were visualized from the load-bearing joints and non-load-bearing xiphoid processes. Significant differences in ciliary morphology were not identified between mutant and wild-type mice. However, after expanding chondrocytes in cell culture and implanting them in solid agarose matrix, it was seen that the fraction of ciliated cells in cultures from mutant mice was significantly lower than in the wild-type cultures (p < 0.05). In addition, in Safranin-O-stained whole joint sections, Bbs mutant mice had significantly lower articular joint thickness (p < 0.05) and lower proteoglycan content saturation (p < 0.05) than wild-type mice. Moreover, there were statistically significant differences of cell distribution between Bbs mutant and wild-type mice (p < 0.05), indicating that mutant articular cartilage had changes consistent with early signs of osteoarthritis. These data indicate that Bbs genes and their functions in the chondrocytic primary cilium are important for normal articular cartilage maintenance.

Fig. 1

Immunofluorescence microscopy of chondrocytic cilia: (a–c) wild-type; (d–f) Bbs6^−/−. Images (a) and (d) display staining with DAPI nuclear marker (blue) and antibody to acetylated α-tubulin (green); (b) and (e) display DAPI with antibody to polaris (red); and (c) and (f) merge all three fluorescent markers. Co-localization of both ciliary proteins, acetylated α-tubulin and polaris, was observed. Bbs1^M390R/M390R and Bbs2^−/− are not shown but demonstrated similar properties.

Fig. 2

Paraffin-embedded tibial plateau sections: (a) wild-type; (b) Bbs1^M390R/M390R; (c) Bbs2^−/−; (d) Bbs6^−/−. Tissues are stained with the proteoglycan marker Safranin-O (red) and Fast Green FCF (blue). Knockin/knockout knees exhibit lower articular cartilage thickness and proteoglycan saturation, along with less mature appearing cell distribution than wild-type knees. 40× magnification insets.

Fig. 3

Histological comparison of tibial plateaus reveals that all knockin/knockout articular cartilage has significantly lower (a) proteoglycan saturation (percent) (p<0.05*) and (b) articular cartilage thickness (µm) (p<0.05*) than wild-type cartilage.

Fig. 4

Cell density analysis of articular cartilage from tibial plateaus: (a) superficial layer (50% of articular cartilage area closest to joint surface); (b) deep layer (50% of articular cartilage area closest to subchondral bone). The superficial layer shows similar cell density in all mouse strains (p>0.05), however, the knockin/knockout cell density in the deep layer is significantly higher than in wild-type cartilage (p<0.05*), indicating a difference in cell distribution.

Fig. 5

Sample immunofluorescence images of chondrocytic cilia in agarose plugs from day 1 of the agarose timecourse: (a) wild-type joints; (b) Bbs2^−/− joints. Images display two-dimensional cross-sectional profiles containing the greatest number of visible cilia in the section; however, serial three-dimensional analysis was completed on all sections. Knockin/knockout cultures exhibit a visibly lower number of ciliated chondrocytes than wild-type cultures. Sections were stained with DAPI nuclear marker (blue) and antibody to acetylated α-tubulin (green).

Fig. 6

Fraction of chondrocytes with visible cilia in agarose timecourse cultures: (a) day 1 xiphoid; (b) day 10 xiphoid; (c) day 1 joints; (d) day 10 joints. In every agarose plug, knockin/knockout cultures had a significantly lower fraction of ciliated cells than wild-type cultures (p<0.05*). The fraction decreased over time from day 1 to 10 in most tissues, however, the knockin/knockout fraction remained significantly lower in both the xiphoid and joints. This analysis was performed by counting whole cells three-dimensionally with serial imaging of each section.