JAWS coordinates chondrogenesis and synovial joint positioning - PubMed (original) (raw)

JAWS coordinates chondrogenesis and synovial joint positioning

Michael L Sohaskey et al. Development. 2008 Jul.

Abstract

Properly positioned synovial joints are crucial to coordinated skeletal movement. Despite their importance for skeletal development and function, the molecular mechanisms that underlie joint positioning are not well understood. We show that mice carrying an insertional mutation in a previously uncharacterized gene, which we have named Jaws (joints abnormal with splitting), die perinatally with striking skeletal defects, including ectopic interphalangeal joints. These ectopic joints develop along the longitudinal axis and persist at birth, suggesting that JAWS is uniquely required for the orientation and consequent positioning of interphalangeal joints within the endochondral skeleton. Jaws mutant mice also exhibit severe chondrodysplasia characterized by delayed and disorganized maturation of growth plate chondrocytes, together with impaired chondroitin sulfation and abnormal metabolism of the chondroitin sulfate proteoglycan aggrecan. Our findings identify JAWS as a key regulator of chondrogenesis and synovial joint positioning required for the restriction of joint formation to discrete stereotyped locations in the embryonic skeleton.

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Figures

Fig. 1

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 WT and _Jaws_−/− embryos (E18.5). (C) Skeletal preparations from WT (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.

Fig. 2

Fig. 2

_Jaws_−/− embryos exhibit delayed chondrocyte maturation. (A) Histological and radioactive in situ hybridization (RISH) 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 delineate the separation of WT prehypertrophic zones, which remain continuous in the _Jaws_−/− embryo. Arrows indicate reduced Ptch1 and Pthrp signals in periarticular chondrocytes. (B) RISH analysis showing delayed terminal hypertrophic chondrocyte differentiation in the _Jaws_−/− humerus (E15.5), evidenced by decreased expression of Mmp13 and Osteopontin. White brackets delineate the separation of WT _Col10a1_-positive hypertrophic zones, which remain continuous in the _Jaws_−/− embryo.

Fig. 3

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 ISH for Gdf5 expression (E14.5). (C) Histological and RISH analysis showing longitudinal expression of joint interzone markers in individual _Jaws_−/− hindlimb digits (E14.5). (D) Exclusion of Bmpr1b RNA and Collagen II 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, WT staining).

Fig. 4

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 (B) and heparan sulfate (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, ΔUA-galNAc; C4S, ΔUA-galNAc4S; C6S, ΔUA-galNAc6S; glcNAc, N-acetylglucosamine; GlcN, ΔUA-glcNAc; GlcNS, ΔUA-glcNS; GlcN(6S), ΔUA-glcNAc6S. *, P < 0.025; ‡, P < 0.0001, _Jaws_−/− vs. WT percentage (n = 5 pairs per genotype). (D) Comparable amounts (mean and s.d.) of total glycosaminoglycans in WT and _Jaws_−/− hindlimbs, normalized to wet tissue weight. Similar results were obtained for forelimbs. n.d., not determined. (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).

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