Fig 1.
Mice TMJ enthesis lacks fibrocartilage.
(A) The craniofacial region in mice (yellow circle): TMJ). (B–D) CT images of mice TMJ. The TMJ condyle has a mushroom-like structure. The pterygoid fovea connects to the lateral pterygoid muscle (LPM). (E–H) Azan staining of the TMJ enthesis. F-H are high-magnification views of the squares in E. Dotted lines in B and C show the section plane of the histological images in E–H. Intramuscular tendons (G points) and extramuscular tendons (F points) extend from the pterygoid fovea through fibrous entheses (F, G arrows). The lower part of the LPM is attached to the pterygoid fovea via periosteum without tendons (arrow in H). LPM: lateral pterygoid muscle, CH: condylar head.
Fig 2.
The TMJ enthesis faces intramembranous bone.
(A–C) Azan staining of the TMJ and surrounding structures. (D–F) Dual-staining development of the TMJ for alkaline phosphatase (ALP) and desmin. (G–I) Schematic representations of the developing TMJ. The black arrows point to the upper margin of the LPM in panels A–I. (J) The skull base angle relative to the LPM’s long axis progressively decreases from E14 to E18. (K–L) Development of the TMJ enthesis from E14.4 to E15.5. By E15.5, the membranous bone is seen posterior to the TMJ enthesis (arrow tips), to which the TMJ enthesis attaches. (M–N) Jaw movements were observed in 22.2% of mice at E16, but in 100.0% at E18 (n = 7 mice per group). (N) Serial photos of jaw movement at E16. (O, P) In situ hybridization of Scx. The TMJ enthesis expresses Scx in the fetal period. (Q) The gap between muscle and bone in the TMJ narrows progressively from E14 to E16 (n = 4 mice per group; Tukey’s multiple comparison test post one-way ANOVA). LPM: lateral pterygoid muscle, CH: condylar head, SP: sphenoid bone, Asterisk: TMJ enthesis.
Fig 3.
Transcription factor SOX9 is downregulated over time in the TMJ enthesis.
(A–D) Sox9CreERT2;R26tdTomato/+ reporter mice. We detected tdTomato+ cells (white arrows) at the enthesis of mice induced at E13, but not any tdTomato+ cells in mice induced at E15 (n = 3 mice per group; unpaired Student’s t-test). (E–J) Immunohistochemical staining for SOX9 and RUNX2. While SOX9 is expressed in the enthesis at E16, this transcription factor is markedly reduced by E18 (n = 3 mice per group, Tukey’s multiple comparison test post one-way repeated measures ANOVA). RUNX2 is expressed in TMJ entheses at E16, with a significant reduction by E18 (n = 3 mice per group, Tukey’s multiple comparison test post one-way repeated measures ANOVA). (K–L) BrdU staining for identifying proliferating cells in the TMJ enthesis. Cells with proliferative activity at E16 and E17 are significantly decreased compared to E15 (n = 5 mice per group, Tukey’s multiple comparison test post one-way ANOVA; repeated measures). (M) mRNA expression at the muscle-bone attachment site (including enthesis). This site at E15 shows higher levels of expression of the Runx2 and Sox9 genes than those at E18 (n = 6 mice per group, unpaired Student’s t-test). (N) mRNA expression in the condyle alone. Runx2 expression is significantly higher at E14 than at E18. The level of Sox9 expression is significantly lower at E14 than at E18 (n = 6 mice per group, unpaired Student’s t-test). LPM: lateral pterygoid muscle, CH: condylar head. SP: sphenoid.
Fig 4.
Wnt1Cre;Sox9flox/+ mice exhibit deformation of the TMJ entheses.
(A–D) Wnt1Cre;R26LacZ/+ mice. The TMJ enthesis expresses β-galactosidase. (E–K) Wnt1Cre;Sox9flox/+ mice. Fluorescence intensity analysis reveals that RUNX2 expression is significantly higher in Wnt1Cre;Sox9flox/+ mice compared to control mice (F-H) (n = with 3 mice per group, unpaired Student’s t-test). Some RUNX2+ cells are found in the LPM of Wnt1Cre;Sox9flox/+ mice (I-K)(n = with 3 mice per group, unpaired Student’s t-test). LPM: lateral pterygoid muscle, CH: condylar head, Asterisk: TMJ enthesis.
Fig 5.
SOX9 downregulation were identified in the enthesis in contact with the clavicle.
(A) Shoulder: Attachment of the supraspinatus muscle to the humerus and attachment of the deltoid muscle to the clavicle. (b)–(d) represent the same magnification. A SOX9+ enthesis is apparent between the supraspinatus muscle and the humerus, whereas no SOX9 expression is found in the enthesis between the deltoid muscle and the clavicle ((a)–(e)). At E13.5, Sox9 is expressed in the deltoid muscle enthesis ((f)–(i)). (B) Elbow: Attachment of the brachial triceps muscle to the ulna. (a)–(d) ((e)–(h)) are at the same magnification. (a’) is the same magnification as (b’). In E15.5 and 16.5, Sox9+ is observed in the elbow enthesis ((a)–(h) and (a’)–(b’)). At E15.5, SOX9+ cell morphology in the tendon attachment region presents as two types: flattened (blue arrowheads), showing polarity like tendon tissues, and elliptical (ovals) ((d)). At E16.5, elliptically shaped Sox9+ cells can be found between cartilage and muscle ((a’)–(b’); ovals). The tissue attachment region to cartilage consists of flat Sox9+ cells with a polarity akin to tendon cells ((a’); blue arrowheads). Bi: biceps brachii muscle, CL: clavicle, De: Deltoid muscle, H: Huemuls, M: medial head of the triceps brachii muscle, L: lateral head of the triceps brachii muscle, asterisk: tendon of the triceps brachii muscle. U: ulna.