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. 2009 Oct 27;106(43):18297-302.
doi: 10.1073/pnas.0908836106. Epub 2009 Oct 8.

Temporomandibular joint formation requires two distinct hedgehog-dependent steps

Patricia Purcell  1 , Brian W Joo, Jimmy K Hu, Pamela V Tran, Monica L Calicchio, Daniel J O'Connell, Richard L Maas, Clifford J Tabin
Affiliations

Temporomandibular joint formation requires two distinct hedgehog-dependent steps

Patricia Purcell et al. Proc Natl Acad Sci U S A. .

Abstract

We conducted a genetic analysis of the developing temporo-mandibular or temporomandi-bular joint (TMJ), a highly specialized synovial joint that permits movement and function of the mammalian jaw. First, we used laser capture microdissection to perform a genome-wide expression analysis of each of its developing components. The expression patterns of genes identified in this screen were examined in the TMJ and compared with those of other synovial joints, including the shoulder and the hip joints. Striking differences were noted, indicating that the TMJ forms via a distinct molecular program. Several components of the hedgehog (Hh) signaling pathway are among the genes identified in the screen, including Gli2, which is expressed specifically in the condyle and in the disk of the developing TMJ. We found that mice deficient in Gli2 display aberrant TMJ development such that the condyle loses its growth-plate-like cellular organization and no disk is formed. In addition, we used a conditional strategy to remove Smo, a positive effector of the Hh signaling pathway, from chondrocyte progenitors. This cell autonomous loss of Hh signaling allows for disk formation, but the resulting structure fails to separate from the condyle. Thus, these experiments establish that Hh signaling acts at two distinct steps in disk morphogenesis, condyle initiation, and disk-condyle separation and provide a molecular framework for future studies of the TMJ.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
RNA expression of Fgfr1, Fgfr2, Fgfr3, Sox5, Sox6, Sox9, Zfp445, Wnt6, and BMP7 in mouse embryonic temporomandibular joint (TMJ). (A–I) In situ hybridization was performed on serial coronal cryosections of E16.5 TMJ. At this developmental stage, the mandibular condyle, temporal fossa, and TMJ disk are morphologically well defined. (Upper right) Different cell zones in the condyle. (Lower right) An in situ hybridization with Collagen X (ColX), a marker for hypertrophic chondrocytes. f, glenoid fossa of the temporal bone; c, condylar head of the mandible; d, joint disk; pe, perichondrium; fl, fibrous cell layer; pl, progenitor cell layer; fc, flattened chondrocytes; hc, hypertrophic chondrocytes.
Fig. 2.
Fig. 2.
RNA expression of Ihh, Ptc1, Smo, Gli1, Gli2, and Gli3 in the mouse temporomandibular joint (TMJ). (A–F) Coronal cryosections of the TMJ at E16.5. In situ hybridization was performed for the indicated genes. Note high Gli2 expression in disk joint. Ihh, Indian hedgehog; Ptc1, Patched 1; Smo, Smoothened; f, glenoid fossa of the temporal bone; c, condylar head of the mandible; d, joint disk.
Fig. 3.
Fig. 3.
Comparison of gene expression patterns between the temporomandibular joint (TMJ) and other developmentally matched synovial joints. Coronal sections of E16.5 TMJ and E14.5 limb (sagittal sections), shoulder (gleno-humeral), and hip joints (ilio-femoral). (A–C) Gdf5, Gdf6, and Gdf7 in situ hybridization. Signal in limb was detected after 2 days, TMJ sections were left for 5 days, and no signal could be detected. Gli1 (D), Gli2 (E), Ihh (F), Sox5 (G), Sox6 (H), Sox9 (I), and Zfp445 (J). Arrowheads mark the location of each joint. f, glenoid fossa of the temporal bone; c, condylar head of the mandible; d, joint disk; w, wrist; di, digits; s, scapula; h, humerus; i, iliac bone; fe, femur.
Fig. 4.
Fig. 4.
Histological analysis of temporomandibular joint (TMJ) abnormalities in Gli2−/− embryos. (A–D) Serial coronal cryosections of E16.5 and E18.5 wild-type (A, C) and mutant mice (B, D). (A) Representative section through an E16.5 wild-type TMJ showing a well-defined condyle, glenoid fossa, and joint disk and distinguishable upper and lower synovial joint cavities. In the condyle, cells are smaller and in higher density in the apical region of the condyle and gradually increase in size and decrease in density toward the lower margin of the condyle. (B) Section through an E16.5 Gli2−/− TMJ. The condyle and fossa can be distinguished clearly, but no joint disk is noticeable. In addition, cells in the condyle are not organized as described in the wild-type littermate (A). (C) Section through an E18.5 wild-type TMJ showing well-defined condyle, glenoid fossa, and fibrous compact joint disk. Upper and lower joint cavities have enlarged. In the condyle, small cells are concentrated in the apical region, and the number of enlarged cells has increased. The perichondrium surrounding the condyle is wider and well defined. (D) Section through the TMJ of an E18.5 Gli2−/− mutant. Condyle, fossa, and upper joint cavity are evidently formed; however, the TMJ disk and lower joint cavity are not visible. Cells in the condyle appear disorganized, and the perichondrium is much thinner. Arrowheads in C and D indicate articular cartilage. uc, upper joint cavity; lc, lower joint cavity.
Fig. 5.
Fig. 5.
Alterations in gene expression in Gli2−/− mouse temporomandibular joint (TMJ). (A–V) Serial coronal cryosections of the TMJ at E18.5 in wild-type and mutant (Gli2−/−) mice. In situ hybridization was performed for the indicated genes. (A, D) Fgfr1, (B, E) Fgfr2, (C, F) Fgfr3, (G, J) Collagen X (ColX), (H, K) Zinc finger protein 445 (Zfp445), (I, L) Sox9, (M, P) Indian hedgehog (Ihh), (N, Q) Patched 1 (Ptc1), (O, R) Patched 2 (Ptc2), (S, U) Gli1, and (T, V) Gli3. f, glenoid fossa of the temporal bone; c, condylar head of the mandible; d, disk. Dashed lines demarcate the boundaries of condyle and fossa.
Fig. 6.
Fig. 6.
Conditional ablation of Smo from chondroprogenitor cells. Cre recombinase activity in Sox9::Cre mice, using the GFP Cre reporter. The green GFP signal indicates cells in which Cre-Lox recombination has taken place, and the red cells represent expression of mCherry where Cre recombination did not occur. GFP and mCherry expression are mutually exclusive. (A) Sox9-expressing chondroprogenitor cells up to E17.5 are GFP-positive. Chondroprogenitor cells are distributed throughout the temporomandibular joint fossa, disk, and condyle. (B) GFP signal in an E17.5 Sox9::Cre; Smofl/fl;RC::Epe compound embryo. Smo was conditionally removed from Sox9-expressing cells. As a result, the condyle is much smaller than its wild-type counterpart, and the lower joint cavity (lc) is absent in the mutant (A, B). (C, D) Hematoxylin and eosin staining at E18.5 to visualize the loss of growth-plate-like organization in the condyle of the mutant (D) compared with its wild-type counterpart (C). uc, upper joint cavity.
Fig. 7.
Fig. 7.
Model for the requirement of hedgehog in temporomandibular joint formation. Indian hedgehog (Ihh) is localized in prehypertrophic chondrocytes in the condyle and required for disk induction. In the absence of Gli2, there is no disk (d) induction. Disk separation and lower joint cavity (lc) formation are inhibited after the removal of Smo from Sox9-expressing cells. uc, upper joint cavity.
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