The postnatal role of Sox9 in cartilage

Abstract

Sox9 is an essential transcription factor for the differentiation of the chondrocytic lineage during embryonic development. To test whether Sox9 continues to play a critical role in cartilaginous tissues in the adult mice, we used an inducible, genetic strategy to disrupt the Sox9 gene postnatally in these tissues. The postnatal inactivation of Sox9 led to stunted growth characterized by decreased proliferation, increased cell death, and dedifferentiation of growth plate chondrocytes. Upon postnatal Sox9 inactivation in the articular cartilage, the sulfated proteoglycan and aggrecan content of the uncalcified cartilage were rapidly depleted and the degradation of aggrecan was accompanied by higher ADAMTS5 immunostaining and increased detection of the aggrecan neoepitope, NITEGE. In spite of the severe loss of Collagen 2a1 mRNA, the Collagen II protein persisted in the articular cartilage, and no histopathological signs of osteoarthritis were observed. The homeostasis of the intervertebral disk (IVD) was dramatically altered upon Sox9 depletion, resulting in disk compression and subsequent degeneration. Inactivation of Sox9 in the IVD markedly reduced the expression of several genes encoding extracellular matrix proteins, as well as some of the enzymes responsible for their posttranslational modification. Furthermore, the loss of Sox9 in the IVD decreased the expression of cytokines, cell-surface receptors, and ion channels, suggesting that Sox9 coordinates a large genetic program that is instrumental for the proper homeostasis of the cells contained in the IVD postnatally. Our results indicate that Sox9 has an essential role in the physiological control of cartilaginous tissues in adult mice. © 2012 American Society for Bone and Mineral Research.

Figure 1

Skeletal phenotype of mice in which the Sox9 gene is disrupted during postnatal growth. Faxitron X-ray and microCT of 4-month-old mice injected with tamoxifen at 6 weeks. A) Left: X-ray of control Sox9^flox/flox mouse; right: X-ray of Sox9^Δ/Δ mouse. Arrow points to kyphosis of the cervical region of the spine. B) MicroCT of Sox9^flox/flox femur C) MicroCT of Sox9^Δ/Δ femur. Arrow points to loss of trabecular bone in the epiphysis of femur underneath the primary spongiosa.

Figure 2

Growth plate examined by histology, proliferation, and cell death assays in postnatal Sox9-depleted mice. Panels A-D) 6-week-old tibial-femoral joint stained with Safranin-O/Fast Green from mice injected with tamoxifen at 3 weeks. A) Sox9^flox/flox B) Sox9^Δ/Δ mouse C) Sox9^flox/flox insert D) Sox9^Δ/Δ mouse, some cells have flattened morphology, see insert. Panels E,F) BrdU stained section from 4-week-old mouse injected with tamoxifen at 3 weeks. E) Sox9^flox/flox insert F) Sox9^Δ/Δ, arrow points to hypocellularity in prehypertrophic zone Panels G,H) Sections of growth plate stained with TUNEL. G) Sox9^flox/flox H) TUNEL positive cells detected in Sox9^Δ/Δ mouse. I) Bar graph showing number of cells (N) on the Y-axis in a 1000 micron width across the growth plate. BrdU- Sox9^flox/flox N=114, Sox9^Δ/Δ N=33; TUNEL- Sox9^flox/flox N=0, Sox9^Δ/Δ N=144 with standard deviation. Panels J,K) Indirect immunofluorescent staining of a 5-week-old growth plate section with antibody LC3B from mice injected with tamoxifen at 3 weeks. White arrow points to LC3B ectopic expression in the growth plate in panel K.

Figure 3

Growth plate characterized with indirect immunofluorescence in Sox9 depleted mice. Panels A–N: Growth plate of 25 day-old day animals that were injected previously with tamoxifen at 18 days. Sox9 immunostaining in Sox9^flox/flox (A), and Sox9^Δ/Δ (B). C,D) Merged dual immunofluorescence with nuclear staining Sox9 (red) and extracellular matrix (ECM) staining of Collagen II (green) in Sox9^flox/flox (C) and Sox9^Δ/Δ (D). Collagen IX immunostaining in Sox9^flox/flox (E) and Sox9^Δ/Δ (F). G,H) Merged dual immunofluorescence staining with a Collagen X antibody (red) and DAPI nuclear acid stain (blue) in Sox9^flox/flox (G) and Sox9^Δ/Δ (H). Aggrecan immunostaining in Sox9^flox/flox (I) and Sox9^Δ/Δ (J). Link protein immunostaining in Sox9^flox/flox (K) and Sox9^Δ/Δ (L). ADAMTS5 immunostaining in femoral growth plate with weak expression in reserve zone (arrow) in Sox9^flox/flox (M) and stronger immunostaining in Sox9^Δ/Δ (N). Panels O,P) Histological sections from the growth plate of 21 day-old day animals that were injected previously with tamoxifen at 18 days. Dual indirect immunofluorescence immunostaining with a Collagen Type I antibody (red) and Collagen Type X antibody (green) in Sox9^flox/flox (O) and Sox9^Δ/Δ (P). Ectopic Collagen Type I protein is detected in ECM of prehypertrophic zone in Sox9^Δ/Δ growth plate (arrow, P). Abbreviations: po-primary ossification, gp- growth plate, so- secondary ossification.

Figure 4

Articular cartilage stained with Safranin-O/Fast Green in postnatal Sox9 depleted mice. A,B) Sections from 4-month-old mice injected with tamoxifen at 6 weeks. A) Sox9^flox/flox B) Sox9^Δ/Δ Loss of Saf-O staining in uncalcified articular cartilage above the tidemark (arrow). Panels C–F) Knee sections from 18-month-old mice injected with tam at 6 months. C) Sox9^flox/flox D) Magnified view of articular cartilage Sox9^flox/flox E) Sox9^Δ/Δ F) Magnified view of articular cartilage Sox9^Δ/Δ. Saf-O staining is reduced in the uncalcified articular cartilage above the tidemark (arrow).

Figure 5

Articular cartilage characterized with immunohistochemistry and indirect immunofluorescence in postnatal Sox9 depleted mice. A–D) Sections of the articular cartilage from 5-month-old mice injected with tam. at 2 months. A) Sox9 immunostaining in uncalcified cartilage above the tidemark Sox9^flox/flox B) Loss of Sox9 immunostaining in articular cartilage Sox9^Δ/Δ C,D)- Dual indirect immunofluorescence with a Sox9 antibody (red) and a Collagen Type II antibody (green) of Sox9^flox/flox (C) and Sox9^Δ/Δ mouse (D). E,F) Sections of the articular cartilage from 9-week-old mice injected with tam. at 8 weeks E) Aggrecan immunostaining in the territorial ECM of Sox9^flox/flox mouse F) Aggrecan levels in the territorial matrix reduced, Sox9^Δ/Δ Panels G–L) Sections of the knee joint from 5 month-old mice injected with tam at 3 months. Aggrecan immunostaining Sox9^flox/flox (G) and Sox9^Δ/Δ mouse (H) Aggrecan is lost from the surface articular cartilage above the tidemark. ADAMTS5 immunostaining Sox9^flox/flox (I) and stronger ADAMTS5 immunostaining Sox9^Δ/Δ (J). Immunostaining with an anti-aggrecan neoepitope antibody, NITEGE, in Sox9^flox/flox (K) and Sox9^Δ/Δ (L). Panels M–P) Sections of the knee joint from 5-month-old mice injected with tamoxifen at 3 months. and subjected to indirect immunofluorescence with an anti-decorin antibody. M) Weak decorin immunostaining surface articular cartilage Sox9^flox/flox mouse N) Strong Decorin immunostaining in Sox9^Δ/Δ articular cartilage. Lubricin immunostaining intensity is similar in Sox9^flox/flox (O) and Sox9^Δ/Δ (P).

Figure 6

Vertebral column of adult Sox9 depleted mice. Spines from 6-month-old animals injected with tam. at 4 months. A) Faxitron of Sox9^flox/flox mouse (left) and Sox9^Δ/Δ mouse (right). Arrow points to an empty space visualized in the X-ray, and presumably the intervertebral disc (IVD). B,C) MicroCT of the lumbar vertebrae 3 and 4 (L3/L4) in the Sox9^flox/flox mouse (B) and Sox9^Δ/Δ mouse (C). The IVD (L3/L4) is depicted as the empty space shown with a red arrow (top) and white arrow (bottom). Compression of IVD Sox9^Δ/Δ D) Von Kossa staining of L4/L5 vertebra in Sox9^flox/flox mouse. Black arrows point to vertebrae growth plate E) Von Kossa staining of lumbar 4/5 vertebra in Sox9^Δ/Δ mouse.

Figure 7

Histological sections of lumbar intervertebral disc. A–D) IVD from 3 month-old injected with tam. at 2 months. A) Indirect immunofluorescence with anti-Sox9 antibody Sox9^flox/flox B) Sox9 antibody (red nuclear) and Collagen Type II antibody (green ECM) of Sox9^flox/flox C) Sox9 immunostaining, Sox9^Δ/Δ D) Sox9 antibody (red) and Collagen Type II antibody (green), Sox9^Δ/Δ mouse. E,F) Saf-O/Fast Green sections 10 week-old injected with tam. at 8 weeks. E) Sox9^flox/flox F) Sox9^Δ/Δ G,H) Saf-O/Fast Green sections 12 week-old injected with tam. at 8 weeks G) Sox9^flox/flox H) Sox9^Δ/Δ I,J) Immunohistochemistry with anti-aggrecan antibody of 10 week-old mice injected with tam. 8 weeks I) Sox9^flox/flox J) Sox9^Δ/Δ Aggrecan depletion in IVD, but aggrecan detected in growth plate, correlating with the Saf-O levels observed in G,H. Panels K,L) Saf-O/Fast Green 8 month-old mice injected with tam. at 6 months of age. K) Sox9^flox/flox L) Sox9^Δ/Δ