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First published online 3 August 2005
doi: 10.1242/dev.01948

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Maintenance of chondroitin sulfation balance by chondroitin-4-sulfotransferase 1 is required for chondrocyte development and growth factor signaling during cartilage morphogenesis

¹ Programme in Molecular Biology and Cancer, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, 600 University Avenue, Toronto, Ontario M5G 1X5, Canada
² The Hope Heart Program, Benaroya Research Institute at Virginia Mason, 1124 Columbia Street, Seattle, WA 98104-2046, USA
³ Division of Cardiovascular Research, The Hospital for Sick Children, Toronto, Ontario M5G 1X8, Canada

View larger version (48K): [in a new window]   Fig. 1. Gene trap integration into the C4st1 locus. (A) Schematic representation of the integration of the PT-1 gene trap vector into intron 1 of the mouse C4st1 locus and the formation of a C4st1-exonI-lacZ fusion transcript. (B) Embryos from intercrosses of C4st1gt heterozygous animals were genotyped by Southern analysis and the ratio of lacZ to en-2 alleles measured. (C-F) Whole-mount in situ hybridization on E10.0 embryos using the indicated exon-specific C4st1 probes. In branchial arches (white arrows) and AER of limb buds (black arrowheads) in C4st1 mutant (gt/gt) embryos, a C4st1 exon I-specific signal (E) is present, but an exon II/III-specific signal (F) is absent.

View larger version (81K): [in a new window]   Fig. 2. Phenotype of the C4st1gt mutation at E19.5 of embryogenesis. (A) Gross morphology of wild-type (part i, +/+) and C4st1gt/gt embryos (ii, part gt/gt). (B-H) Alcian blue/Alizarin Red skeletal stains. (B) Multiple skeletal abnormalities are evident in mutant embryos. (C) Higher magnification of hind limbs, showing the severely shortened and thickened iliac bone (i), femur (f) and tibia and fibula (ti). Arrowhead indicates Alcian Blue staining of cartilage. (D) Vertebrae in the mutant display misshapen dorsal arches (arrowhead). (E) Reduced size of scapula (double-headed while arrow) in mutant embryos. (F) Phalanges 2 and 3, and the talus bone (arrow) fail to ossify in mutant hindlimbs (m, metatarsal bones). (G) Lateral view and (H) dorsal view of skull, showing normal size of frontal (a), parietal (b), interparietal (c) and supraoccipital (d) bones in mutant embryos, but smaller maxilla (arrowhead), mandible (arrow) and nasal bones in mutant embryos.

View larger version (79K): [in a new window]   Fig. 3. Analysis of cartilage development in C4st1gt/gt embryos by staining for lacZ. Part i, wild type; part ii, mutant. (A,D) Identical staining in whole-mount (A) and sectioned (D) forelimb buds of cartilage aggregations (arrows) in +/gt (i) and gt/gt (ii) E11.5 embryos. Asterisk indicates AER. (B,E) Whole-mount staining of cartilage primordia (B) and sectioning of stained tibia primordium (E) at E13.5 in +/gt (i) and gt/gt (ii) hindlimbs shows no differences in size of cartilage elements or cellular patterning. Abbreviations in B: d, digits; t, tibia; fi, fibula; f, femur. Abbreviations in E: d, distal; p, proximal. Arrowhead indicates slight bending of gt/gt tibia primordium. (C) Whole-mount staining of hindlimbs at E15.5, showing impaired segmentation of cartilage in digits (arrowhead) and bending of tibia (arrow) in gt/gt (ii), but not +/gt (i) embryos. d, digits; t, tibia; fi, fibula. (F) Sectioned proximal tibia of stained +/gt (i) and gt/gt (ii) E15.5 hindlimbs. Homozygous mutant growth plates are slightly shortened (double-headed arrow) and show a decrease in the size of the columnar zone (c). p, proliferative zone; h, hypertrophic zone. Scale bar: 100 µm for A,D; 300 µm for B; 100 µm for E; 600 µm for C; 200 µm for F.

View larger version (138K): [in a new window]   Fig. 4. Cartilage growth plate defects in the proximal tibia of C4st1gt mutant embryos at E18.5. (A) RNA in situ hybridization using a C4st1-exon2+3-specific probe shows C4st1 expression in proliferating, but not hypertrophic chondrocytes in wild-type growth plates (i) and the reduction in C4st1 staining in mutant growth plates (ii). (B,C) Safranin O staining. (B) Safranin O staining shows a reduction in size of proliferating (p), columnar (c) and hypertrophic (h) zones, as well as less intense staining in mutant growth plates (ii) when compared with wild type (i). Brackets indicate areas shown in C. (C) Higher magnification of the transition zone between hypertrophic cartilage and primary bone, showing cartilage islands (arrow) in wild-type (i), but not mutant, bone (ii). (D-G) Hematoxylin and Eosin staining. (D) Mutant growth plates appear disorganized and contain ECM disruptions (arrowhead) and misoriented chondrocyte columns (arrow). (E,F) Higher magnification either wild-type (E) or mutant (F) growth plates (p, c and h are defined in B). Wild-type chondrocyte columns are oriented parallel to the longitudinal bone axis, whereas mutant columns are oriented almost perpendicular to it. In addition there is an increased thickness of the bone collar in the mutants (arrows). (G) Dark-field images of Hematoxylin and Eosin-stained wild-type (i) and mutant (ii) proliferating chondrocytes, showing fibrillation of ECM in mutant growth plates. Scale bar: 400 µm for A; 1 mm for B; 100 µm for C,D; 30 µm for E,F; 10 µm for G.

View larger version (101K): [in a new window]   Fig. 5. Analysis of growth plate extracellular matrix (ECM) markers of the proximal tibia at E18.5. (A) Fluorophore-assisted carbohydrate electrophoresis (FACE) analysis of ECM glycosaminoglycans (GAGs). (B) Chondroitin sulfate (CS) immunohistochemistry showing ECM staining (black arrow) in all three growth plate layers (p, proliferating chondrocytes; c, columnar chondrocytes; h, hypertrophic chondrocytes) of wild-type cartilage (+/+), whereas CS staining in mutant (gt/gt) proliferating and columnar layers is restricted to the pericellular space (arrowhead), with very little staining in the ECM (black arrow). However, in the columnar and hypertrophic layers of mutants, ECM staining of CS was observed (white arrows). (C) C6S was detected by immunofluorescence staining and is distributed in the outer-most layers of the proliferative zone in wild-type cartilage (+/+), whereas in mutant cartilage (gt/gt), low pericellular C6S staining was observed in both proliferative and columnar layers. (D) Distribution of aggrecan was analyzed by immunofluorescence, which revealed localization to the ECM in all layers of the wild-type cartilage (+/+), as well as in the proliferative layer of mutant cartilage (gt/gt). However, in the mutant, aggrecan was increasingly restricted to the pericellular space in columnar and hypertrophic (arrowhead) layers. (E) Detection of collagen II by immunofluorescence shows strong staining of the ECM in both wild-type (+/+) and mutant (gt/gt) proliferative and columnar layers, and decreased staining in the ECM of the hypertrophic layer. Scale bar: 10 µm.

View larger version (95K): [in a new window]   Fig. 6. Chondrocyte development and differentiation at E18.5. (A,B,G-I) Proximal tibia; (C-F) distal tibia. (A-C) Marker analysis by RNA in situ hybridization in wild-type (+/+) and C4st1gt/gt (gt/gt) growth plates. Developmental markers for proliferating chondrocytes (collagen II, A), hypertrophic chondrocytes (collagen X, B) as well as columnar chondrocytes (Fgfr3, C) were expressed normally in mutant growth plates (ii) when compared with their wild-type counterparts (i), with the size of their expression domains reduced in relation to the overall shortening of the growth plate. (D) BrdU labeling of proliferating chondrocytes was detected by immunofluorescence (green). In wild-type growth plates (i), the zone of proliferation (double-headed arrow) excludes the hypertrophic layer. This proliferative zone is reduced in size in mutant cartilage (ii). (E) TUNEL staining (green) to identify chondrocytes undergoing apoptosis. In wild-type cartilage (i), low numbers of only the most differentiated hypertrophic cells (white bar) showed TUNEL staining, whereas in mutant cartilage, an increased number of cells in all zones (double-headed arrow) showed TUNEL staining (ii). (F) Immunofluorescence (red) using an -Bcl2 antibody. Wild-type growth plates (i) show widespread Bcl2 staining in the columnar zone (double-headed arrow), whereas staining in gt/gt growth plates (ii) is severely reduced and present only in lateral areas (arrows). (G) Immunofluorescence (red) using an -Bax antibody. Wild-type growth plates (i) show strong staining in prehypertrophic and hypertrophic chondrocytes (double-headed arrow), but not in columnar and proliferating cells (arrowhead). Homozygous mutant growth plates (ii), however, display staining in hypertrophic cells (arrow) and proliferating cells (arrowhead). (H,I) Higher magnification of regions labeled by white squares in G. Bax expression in wild-type growth plates is restricted to prehypertrophic and hypertrophic cells (`h' in H, i), and is not present in columnar cells (`c' in H, i) or proliferating cells (I, i). White line represents the border between columnar and prehypertrophic and hypertrophic zones. Bax expression in mutant growth plates is observed in hypertrophic cells (arrowhead in H, ii), but also in columnar cells (arrow in H, ii) and proliferating cells (green arrow in H, ii; arrowhead in I, ii). (J) Quantitation of BrdU-labeled cells in proliferating (prol.), columnar (column.) and hypertrophic (hypertr.) chondrocytes. Cartilage in mutant (gt/gt) shows an approximate twofold increase in BrdU-labeled cells in both proliferating and columnar chondrocytes compared with wild type. (K) Quantitation of TUNEL-labeled (apoptotic) cells in all three layers of the growth plate. Mutant growth plates showed a 3.5-fold increase in the number of cells undergoing apoptosis in the hypertrophic zone and apoptotic cells in both proliferating and columnar chondrocytes. Scale bar: 150 µm.

View larger version (135K): [in a new window]   Fig. 7. Analysis of Ihh, BMP and TGFß signaling in E18.5 cartilage. (A) distal tibia; (B-H) proximal tibia. (A) Ptch1 RNA in situ hybridization as output for Ihh signaling reveals expression in columnar chondrocytes in both wild-type (i) and mutant (ii) cartilage. (B-D) Phosphorylated Smad1 distribution. Wild-type (i) and mutant (ii) growth plates were stained using an antibody that recognizes Smad1 phosphorylated by activated BMP receptors. (B) pSmad1 (red) is seen in hypertrophic chondrocytes in wild-type cartilage (i), and is reduced in mutant cartilage (ii). (C) Higher magnification of proliferating region (indicated as `C' in B) shows very little pSmad1 staining in both wild-type (i) and mutant (ii) cartilage. (D) Higher magnification of early hypertrophic regions (indicated as `D' in B) shows nuclear pSmad1 staining in wild-type chondrocytes (i), which is reduced in mutant chondrocytes (ii). (E-H) Phosphorylated Smad2 distribution. Wild-type and mutant growth plates were stained using an antibody that recognizes Smad2 phosphorylated by activated TGFß receptors. (E) Very little nuclear pSmad2 staining was seen in wild-type growth plates (i) in columnar/early hypertrophic layers and in lateral aspects of the growth plate (white arrow). In mutant cartilage (ii), strong pSmad2 staining is seen in all cartilage layers. (F-H) Higher magnification of regions indicated in E. Arrows indicate nuclear staining. (F) Higher magnification of proliferative layer. No nuclear pSmad2 staining was seen in wild-type chondrocytes (i), whereas a high proportion of mutant chondrocytes (ii) shows moderate nuclear pSmad2 staining. (G) Higher magnification of late columnar layer. Very little nuclear pSmad2 staining is visible in wild-type cells (i), whereas strong nuclear pSmad2 staining is apparent in all mutant chondrocytes (ii). (H) Higher magnification of hypertrophic layers. Weak nuclear pSmad2 staining is seen in few wild-type chondrocytes (i), whereas almost all mutant chondrocytes show strong nuclear pSmad2 staining (ii). Scale bar: 300 µm for A; 200 µm for B,E; 20 µm for C,D,F,G,H.

View larger version (75K): [in a new window]   Fig. 8. C4st1gt/gt metatarsal explants are able to respond to exogenous growth factors. Metatarsals were removed from E18.5 wild-type (+/+) and mutant (gt/gt) embryos and cultures for 4 days in either the absence (control) or presence of 2 µg/ml N-Shh, 500 pM TGFß or 10nM BMP2 as indicated. Metatarsals were photographed (A,B) or processed for RNA in situ hybridization (C-F) or immunofluorescence (G-J). (A,B) Appearance of explants after 4 day treatment with no factor (control), N-Shh, BMP2 or TGFß. (C,D) Effects of growth factor treatment on hypertrophic differentiation as visualized by collagen X RNA in situ hybridization. Collagen X staining is reduced in N-Shh and TGFß-treated wild-type (C) and mutant (D) explants and increased in BMP2-treated wild-type and mutant explants. In addition, treatment of wild-type, but not mutant explants with N-Shh lead to increased thickness of the perichondrium (arrows). (E-J) Signaling pathways in metatarsal explants. (E) Ptch1 staining in untreated wild-type and mutant explants is restricted to proliferating chondrocytes. No Ptch1 expression is seen in the perichondrium. (F) Treatment of explants with N-Shh leads to Ptch1 expression throughout the growth plate in both wild-type and mutant explants. Wild-type explants also showed Ptch1 expression in the perichondrium, which was not observed in mutant explants (arrows). (G) Nuclear pSmad2 staining in untreated wild-type explants was seen in some late columnar/early hypertrophic cells (i; see iv for higher magnification), whereas occasional weak staining in proliferating cells was also present (i; iii). In untreated mutant explants, strong pSmad2 staining was apparent in all cells of the growth plate (ii, v, vi). (H) Treatment of wild-type explants with TGFß lead to an increase in the number of pSmad2-stained cells and staining intensity in hypertrophic cells (i, iv), whereas cells in the proliferative layer were still mostly negative for nuclear pSmad2 (i, iii). Arrow in i indicates increased pSmad2 staining in lateral regions of the growth plate. Treatment of mutant explants with TGFß lead to a small increase in pSmad2 staining intensity (ii, v, vi). (I) Nuclear pSmad1 expression in untreated wild-type explants was apparent in a subset of proliferating chondrocytes (iii) and in hypertrophic chondrocytes (iv). Whereas mutant explants also showed nuclear pSmad1 staining in hypertrophic chondrocytes (ii, vi), staining intensity was lower in proliferating chondrocytes (ii, v). (J) Both wild-type (i, iii, iv) and mutant (ii, v, vi) explants treated with BMP2 showed strong nuclear pSmad1 staining in the expanded region of hypertrophy (iii, iv, v, vi). Scale bar: 5 mm for A,D; 1 mm for C-F; 500 µm for G-J, parts i, ii; 20 µm for G-J, parts iii-vi.

View larger version (51K): [in a new window]   Fig. 9. Model of the role of C4st1 during cartilage development. The organized growth plate of wild-type cartilage contains proliferating (p), columnar (c) and hypertrophic (h) chondrocytes (grey) surrounded by ECM containing CS (orange). C4st1gt/gt-mutant growth plates are severely reduced in length, but display increased width. The chondrocyte layers are disorganized and chondrocyte columns are not oriented along the longitudinal axis of the bone. CS (orange) is mostly absent from the ECM and instead is located in the pericellular space surrounding chondrocytes. There is a dramatic increase in the thickness of the bone collar (dark grey) in mutant growth plates and bone. Whereas Ihh signaling to proliferating chondrocytes is not significantly altered in mutant growth plates (red arrows), BMP signaling to both proliferating and hypertrophic chondrocytes is reduced (yellow arrows), and TGFß signaling to proliferating and hypertrophic chondrocytes (blue arrows) is dramatically increased in mutant growth plates. Imbalance in apoptotic signals leads to a dramatic increase in the area prone to undergo apoptosis (indicated by black brackets).

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