Postnatal lethality and chondrodysplasia in mice lacking both chondroitin sulfate N-acetylgalactosaminyltransferase-1 and -2

Abstract

Chondroitin sulfate (CS) is a sulfated glycosaminoglycan (GAG) chain. In cartilage, CS plays important roles as the main component of the extracellular matrix (ECM), existing as side chains of the major cartilage proteoglycan, aggrecan. Six glycosyltransferases are known to coordinately synthesize the backbone structure of CS; however, their in vivo synthetic mechanism remains unknown. Previous studies have suggested that two glycosyltransferases, Csgalnact1 (t1) and Csgalnact2 (t2), are critical for initiation of CS synthesis in vitro. Indeed, t1 single knockout mice (t1 KO) exhibit slight dwarfism and a reduction in CS content in cartilage compared with wild-type (WT) mice. To reveal the synergetic roles of t1 and t2 in CS synthesis in vivo, we generated systemic single and double knockout (DKO) mice and cartilage-specific t1 and t2 double knockout (Col2-DKO) mice. DKO mice exhibited postnatal lethality, whereas t2 KO mice showed normal size and skeletal development. Col2-DKO mice survived to adulthood and showed severe dwarfism compared with t1 KO mice. Histological analysis of epiphyseal cartilage from Col2-DKO mice revealed disrupted endochondral ossification, characterized by drastic GAG reduction in the ECM. Moreover, DKO cartilage had reduced chondrocyte proliferation and an increased number of apoptotic chondrocytes compared with WT cartilage. Conversely, primary chondrocyte cultures from Col2-DKO knee cartilage had the same proliferation rate as WT chondrocytes and low GAG expression levels, indicating that the chondrocytes themselves had an intact proliferative ability. Quantitative RT-PCR analysis of E18.5 cartilage showed that the expression levels of Col2a1 and Ptch1 transcripts tended to decrease in DKO compared with those in WT mice. The CS content in DKO cartilage was decreased compared with that in t1 KO cartilage but was not completely absent. These results suggest that aberrant ECM caused by CS reduction disrupted endochondral ossification. Overall, we propose that both t1 and t2 are necessary for CS synthesis and normal chondrocyte differentiation but are not sufficient for all CS synthesis in cartilage.

Fig 1. Phenotype of t2 null mice.

(A) Schematic showing the CS biosynthetic pathway and relevant glycosyltransferases. Arrows indicate the catalytic activity of each glycosyltransferase. Half-filled diamonds, open squares, open circles, and stars refer to GlcA, GalNAc, Gal, and Xyl, respectively. (B) Quantitative analysis of t1 and t2 gene transcription in humeral cartilage of WT mice (n = 3) using real-time RT-PCR. The expression of each transcript was normalized to that of β-actin. (C) Targeting strategy for conditional deletion of the t2 gene. The exon containing the initiation codon and transmembrane domain was flanked by loxP elements. This region was deleted via mating with Ayu1-Cre mice, to generate systemic t2 KO mice. Probe position for Southern hybridization is indicated by the bold line. (D) Southern blot analysis of Bgl II-digested genomic DNA and PCR-based genotyping of progeny from intercrossing heterozygotes. (E) Postnatal growth kinetics of WT mice (male, n = 3; female, n = 6) and t2 KO littermates (male, n = 11; female, n = 5). F-H, Double whole body staining with Alizarin red and Alcian blue (F), upper (G) and lower limbs (H) of the WT and t2 KO littermates at E18.5. Scale bars: 1 cm (F) and 1 mm (G, H).

Fig 2. Phenotype of DKO mice.

(A) Representative photograph of t2 KO pups and cyanotic DKO littermates at E18.5. Scale bar: 1 cm. (B) Body weight of t2 KO (n = 68) and DKO (n = 51) embryos at E18.5. *P < 0.05. (C) Lung sections from t2 KO embryos and DKO littermates were stained with HE. Scale bar: 100 μm. (D-H) Skeletal whole body (D), upper limb (E), lower limb (F), lumber spine (G), and cranial bone (H) preparation. (I) Humeral and tibial bone length at E18.5 of t2 KO (n = 5) and DKO (n = 10) mice. **P < 0.01.

Fig 3. Phenotype of Col2-DKO mice.

(A) Representative photograph of Control pups and Col2-DKO littermates at E18.5, P7, and P14. Scale bar: 1 cm. (B) Body weight of Control (E18.5; n = 16, P7; n = 4, P14; n = 4) and Col2-DKO (E18.5; n = 12, P7; n = 4, P14; n = 4) mice at various stages. *P < 0.05, **P < 0.01. (C) Humeral and tibial bone length at E18.5 of Control (n = 6) and Col2-DKO (n = 7) mice. (D) Quantitative analysis of t1 and t2 transcripts in the knee cartilage of WT (n = 3) and Col2-DKO (n = 3) mice at E18.5 using real-time RT-PCR. The expression of each transcript was normalized to that of β-actin. **P < 0.01.

Fig 4. Safranin-O staining, CS content, and immunohistochemical analyses in cartilage.

(A) Safranin-O staining in E18.5 proximal tibial cartilage of WT, t1 KO, t2 KO, DKO, and Col2-DKO mice. (B) Safranin-O staining in P14 tibial cartilage of Control and Col2-DKO mice. Arrows indicate secondary ossification centers. Each right panel is a higher magnification image of the regions labeled with white squares in the left panel. Black arrowheads indicate abnormal shape and cell layer structure of the growth plate in Col2-DKO mice. White arrowheads indicate ectopic localization of proliferating chondrocytes in the hypertrophic zone. (C) The total amount and disaccharide analysis of the rib cartilage in WT, t1 KO, t2 KO and DKO mice. Δdi-0S, ΔHexAα1-3GalNAc; Δdi-4S, ΔHexAα1-3GalNAc(4S); Δdi-6S, ΔHexAα1-3GalNAc(6S). (D) Immunohistochemical aggrecan staining using 1C6 monoclonal antibody. The sections from Control and Col2-DKO mice were used for staining after chondroitinase ABC treatment. Each lower panel shows a higher magnification image of the regions labeled by magenta squares in the upper panel. Scale bar: 100 μm. (E) Immunostaining with an antibody against type X collagen in Control and Col2-DKO cartilage at E18.5 and P14. P, Proliferative chondrocyte; H, Hypertrophic chondrocyte. Scale bar: 100 μm.

Fig 5. Cell proliferation and apoptosis in proximal tibial epiphyseal cartilage.

(A) Quantification of cell proliferation assessed by Ki67 immunostaining in tibia sections from Control (E18.5; n = 4, P14; n = 3) and Col2-DKO (E18.5; n = 3, P14; n = 6) mice at P14. **P < 0.01. (B) TUNEL staining of tibia sections from Control (n = 3) and Col2-DKO (n = 3) mice at E18.5 and P14. P, Proliferative chondrocyte; H, Hypertrophic chondrocyte; B, Bone. Scale bar: 100 μm.

Fig 6. GAG accumulation and cell proliferation in primary chondrocyte cultures.

(A) Proliferation of WT (n = 3) and Col2-DKO (n = 3) chondrocytes. (B) Primary chondrocytes from knee cartilage of WT and Col2-DKO mice at E18.5 were stained for GAG accumulation using Alcian blue at culture day 7. (C) The absorption intensity at 595 nm of Alcian blue-stained cell extracts in WT (n = 3) and Col2-DKO (n = 3) mice was measured to quantify sulfated GAG accumulation. *P < 0.05.

Fig 7. Quantitative gene expression and in situ hybridization of genes associated with endochondral ossification.

(A) Quantitative analysis of transcription of ECM and Ihh signaling molecules in E18.5 humeral cartilage using real-time RT-PCR. The expression of each transcript was normalized to that of Hprt. The amount of each transcript in WT cartilage was set to a value of 1.0. *P < 0.05. (B) The expression of the indicated probes was examined by in situ hybridization in tibial sections at E18.5. The black dotted lines indicate the contour of the proximal epiphysis of the tibia. Scale bar: 100 μm.