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First published online June 6, 2008
doi: 10.1242/10.1242/dev.019950

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JAWS coordinates chondrogenesis and synovial joint positioning

¹ Division of Genetics, Genomics and Development, Department of Molecular and Cell Biology, Center for Integrative Genomics, University of California, Berkeley, CA 94720, USA.
² Departments of Internal Medicine (Rheumatology) and Biochemistry, Rush University Medical Center, 1725 Harrison Street, Chicago, IL 60612, USA.

View larger version (59K): [in this window] [in a new window]   Fig. 1. Insertional mutagenesis of Jaws causes chondrodysplasia. (A) Widespread β-galactosidase staining from the gene trap reporter in Jaws+/- E12.5 forelimb (left) and E15.5 digits (right). (B) Gross morphology (top) and cleared skeletal preparations (bottom) of wild-type (WT) and Jaws-/- embryos (E18.5). (C) Skeletal preparations from wild-type (top or left) and Jaws-/- (bottom or right) embryos, showing severe malformation of endochondral elements. (D) Alcian Blue/von Kossa staining of humerus sections (E17.5). The arrow indicates ectopic chondrocytes encroaching upon the primary ossification center. Higher-magnification views (top) show hypocellularity and ECM discontinuity in the Jaws-/- growth plate.

View larger version (62K): [in this window] [in a new window]   Fig. 2. Jaws-/- embryos exhibit delayed chondrocyte maturation. (A) Histological and radioactive in situ hybridization analysis of chondrocyte maturation in the humerus (E14.5). Top panels show sections stained with Alcian Blue and nuclear Fast Red (AB/NFR). White brackets indicate the separation of wild-type prehypertrophic zones, which remain continuous in the Jaws-/- embryo (vertical lines). Arrows indicate reduced Ptch1 and Pthrp signals in periarticular chondrocytes. (B) Radioactive in situ hybridization analysis showing delayed terminal hypertrophic chondrocyte differentiation in the Jaws-/- humerus (E15.5), evidenced by decreased expression of Mmp13 and osteopontin. White brackets indicate the separation of wild-type Col10a1-positive hypertrophic zones, which remain continuous in the Jaws-/- embryo (vertical line).

View larger version (92K): [in this window] [in a new window]   Fig. 3. Jaws-/- digits develop ectopic joints. (A) Longitudinal cavities lacking Alcian Blue staining form in Jaws-/- digits (E18.5). Higher-magnification views of digit 3 are shown to the right of each limb, with arrows indicating joint position and orientation. The arrowhead indicates failed knee joint formation proximal to a lack of tibial ossification in the Jaws-/- hindlimb. (B) Whole-mount in situ hybridization for Gdf5 expression (E14.5). (C) Histological and in situ hybridization analysis showing longitudinal expression of joint interzone markers in individual Jaws-/- hindlimb digits (E14.5). (D) Exclusion of Bmpr1b RNA and collagen 2 protein expression from ectopic Jaws-/- joints (E14.5). (E) Ectopic joints express Gdf5 but not markers of hypertrophic chondrocytes (Col10a1), osteoblasts (Runx2), or tendons and ligaments (Scx) (E18.5 forelimb digits). AB/vK, Alcian Blue/von Kossa. (F) Defective cavitation of E18.5 ectopic joint (arrow), evidenced by the absence of CD44 immunostaining (arrowhead, wild-type staining).

View larger version (48K): [in this window] [in a new window]   Fig. 4. Impaired chondroitin sulfation and altered aggrecan metabolism in Jaws-/- limbs. (A) CS immunostaining in hindlimb digits (E14.5). Higher-magnification views (top) reveal sparse irregular staining in the Jaws-/- joint. (B,C) Fluorophore-assisted carbohydrate electrophoresis analysis of chondroitin sulfate (CS, B) and heparan sulfate (HS, C) in E14.5 limbs. The relative proportions and percentages of disaccharides generated with the glycosaminoglycan-specific lysases are shown. For HS, no disaccharides containing 2-sulfated uronic acid (UA) or N,6-sulfated GlcN were detected. galNAc, N-acetylgalactosamine; C0S, UA-galNAc; C4S, UA-galNAc4S; C6S, UA-galNAc6S; glcNAc, N-acetylglucosamine; GlcN, UA-glcNAc; GlcNS, UA-glcNS; GlcN(6S), UA-glcNAc6S. *P<0.025; P<0.0001, Jaws-/- versus wild type percentage (n=5 pairs per genotype). (D) Comparable amounts (mean and s.d.) of total glycosaminoglycans in wild type and Jaws-/- hindlimbs, normalized to wet tissue weight. Similar results were obtained for forelimbs. n.d., not determined; HA, hyaluronan. (E) Aggrecan immunostaining in the proximal end of the humerus (E14.5). (F) Anti-CDAGWL immunoblots of E14.5 limb protein extracts, showing less full-length aggrecan (arrow, 350 kDa) and fewer aggrecan cleavage fragments (bracket) in Jaws-/- cartilage. Successive nondissociative extractions (lanes 1 and 2; arrowhead indicates a 65 kDa proteolytic fragment) were followed by one dissociative extraction (lane 3). HL, hindlimbs; FL, forelimbs.

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