An in situ hybridization study of the Syndecan family in the developing condylar cartilage of fetal mouse mandible.

Mandibular condylar cartilage is a representative secondary cartilage, differing from primary cartilage in various ways. Syndecan is a cell-surface heparan sulfate proteoglycan and speculated to be involved in chondrogenesis and osteogenesis. This study aimed to investigate the expression patterns of the syndecan family in the developing mouse mandibular condylar cartilage. At embryonic day (E)13.0 and E14.0, syndecan-1 and -2 mRNAs were expressed in the mesenchymal cell condensation of the condylar anlage. When condylar cartilage was formed at E15.0, syndecan-1 mRNA was expressed in the embryonic zone, wherein the mesenchymal cell condensation is located. Syndecan-2 mRNA was mainly expressed in the perichondrium. At E16.0, syndecan-1 was expressed from fibrous to flattened cell zones and syndecans-2 was expressed in the lower hypertrophic cell zone. Syndecan-3 mRNA was expressed in the condylar anlage at E13.0 and E13.5 but was not expressed in the condylar cartilage at E15.0. It was later expressed in the lower hypertrophic cell zone at E16.0. Syndecan-4 mRNA was expressed in the condylar anlage at E14.0 and the condylar cartilage at E15.0 and E16.0. These findings indicated that syndecans-1 and -2 could be involved in the formation from mesenchymal cell condensation to condylar cartilage. The different expression patterns of the syndecan family in the condylar and limb bud cartilage suggest the functional heterogeneity of chondrocytes in the primary and secondary cartilage.

Circadian BMAL1 regulates mandibular condyle development by hedgehog pathway.

OBJECTIVE: Chondrogenesis and endochondral ossification in mandibular condyle play crucial roles in maxillofacial morphogenesis and function. Circadian regulator brain and muscle arnt-like 1 (BMAL1) is proven to be essential for embryonic and postnatal development. The goal of this study was to define the functions of BMAL1 in the embryonic and postnatal growth of mandibular condylar cartilages (MCC). MATERIALS AND METHODS: Micro-CT, TUNEL staining and EdU assay were performed using BMAL1-deficient mice model, and in vitro experiments were performed using rat chondrocytes isolated from MCC. RNA sequencing in mandibular condyle tissues from Bmal1-/- mice and the age-matched wild-type mice was used for transcriptional profiling at different postnatal stages. RESULTS: The expression levels of BMAL1 decrease gradually in MCC. BMAL1 is proved to regulate sequential chondrocyte differentiation, and its deficiency can result in the impairment of endochondral ossification of MCC. RNA sequencing reveals hedgehog signalling pathway is the potential target of BMAL1. BMAL1 regulates hedgehog signalling and affects its downstream cascades through directly binding to the promoters of Ptch1 and Ihh, modulating targets of hedgehog signalling which is indispensable for endochondral ossification. Importantly, the short stature phenotypes caused by BMAL1 deficiency can be rescued by hedgehog signalling activator. CONCLUSIONS: Collectively, these results indicate that BMAL1 plays critical roles on chondrogenesis and endochondral ossification of MCC, giving a new insight on potential therapeutic strategies for facial dysmorphism.

The Roles of Indian Hedgehog Signaling in TMJ Formation.

The temporomandibular joint (TMJ) is an intricate structure composed of the mandibular condyle, articular disc, and glenoid fossa in the temporal bone. Apical condylar cartilage is classified as a secondary cartilage, is fibrocartilaginous in nature, and is structurally distinct from growth plate and articular cartilage in long bones. Condylar cartilage is organized in distinct cellular layers that include a superficial layer that produces lubricants, a polymorphic/progenitor layer that contains stem/progenitor cells, and underlying layers of flattened and hypertrophic chondrocytes. Uniquely, progenitor cells reside near the articular surface, proliferate, undergo chondrogenesis, and mature into hypertrophic chondrocytes. During the past decades, there has been a growing interest in the molecular mechanisms by which the TMJ develops and acquires its unique structural and functional features. Indian hedgehog (Ihh), which regulates skeletal development including synovial joint formation, also plays pivotal roles in TMJ development and postnatal maintenance. This review provides a description of the many important recent advances in Hedgehog (Hh) signaling in TMJ biology. These include studies that used conventional approaches and those that analyzed the phenotype of tissue-specific mouse mutants lacking Ihh or associated molecules. The recent advances in understanding the molecular mechanism regulating TMJ development are impressive and these findings will have major implications for future translational medicine tools to repair and regenerate TMJ congenital anomalies and acquired diseases, such as degenerative damage in TMJ osteoarthritic conditions.

Part I: Development and Physiology of the Temporomandibular Joint.

PURPOSE OF REVIEW: Investigate the developmental physiology of the temporomandibular joint (TMJ), a unique articulation between the cranium and the mandible. RECENT FINDINGS: Principal regulatory factors for TMJ and disc development are Indian hedgehog (IHH) and bone morphogenetic protein (BMP-2). The mechanism is closely associated with ear morphogenesis. Secondary condylar cartilage emerges as a subperiosteal blastema on the medial surface of the posterior mandible. The condylar articular surface is immunoreactive for tenascin-C, so it is a modified fibrous periosteum with an underlying proliferative zone (cambrium layer) that differentiates into fibrocartilage. The latter cushions high loads and subsequently produces endochondral bone. The TMJ is a heavily loaded joint with three cushioning layers of fibrocartilage in the disc, as well as in subarticular zones in the fossa and mandibular condyle. The periosteal articular surface produces fibrocartilage to resist heavy loads, and has unique healing and adaptive properties for maintaining life support functions under adverse environmental conditions.

The Zonal Architecture of the Mandibular Condyle Requires ADAMTS5.

Temporomandibular joint (TMJ) osteoarthritis (TMJOA) disrupts extracellular matrix (ECM) homeostasis, leading to cartilage degradation. Upregulated a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-5 leads to cleavage of its substrate aggrecan (Acan) and is considered a hallmark of TMJOA. However, most research on ADAMTS5-Acan turnover has focused on hyaline cartilage, not fibrocartilage, which comprises the TMJ. The mandibular condylar cartilage (MCC) of the TMJ is organized in zones, and chondrocytes are arranged in axial rows, yet the molecular mechanisms required to generate the MCC zonal architecture have not been elucidated. Here, we test the hypothesis that ADAMTS5 is required for development of the TMJ MCC. Adamts5+/+ and Adamts5-/- murine TMJs were harvested at postnatal day 7 (P7), P21, 2 mo, and 6 mo of age; histomorphometrics indicated increased ECM. Immunohistochemistry and Western blots demonstrated the expanded ECM correlated with increased Acan localization in Adamts5-/- compared to Adamts5+/+. Cell volume was also decreased in the MCC of Adamts5-/- due to both a reduction in cell size and less mature hypertrophic chondrocytes. Analysis of chondrogenic maturation markers by quantitative real-time polymerase chain reaction indicated Col2a1, Col10a1, and Sox9 were significantly reduced in Adamts5-/- MCC compared to that of Adamts5+/+. The older (6 mo) Adamts5-/- MCC exhibited changes in chondrogenic cell arrangements, including clustering and chondrogenic atrophy, that correlated with early stages of TMJOA using modified Mankin scoring. These data indicate a potentially novel and critical role of ADAMTS5 for maturation of hypertrophic chondrocytes and establishment of the zonal architecture that, when disrupted, may lead to early onset of TMJOA.

An in situ hybridization study of perlecan, DMP1, and MEPE in developing condylar cartilage of the fetal mouse mandible and limb bud cartilage.

The main purpose of this in situ hybridization study was to investigate mRNA expression of three bone/cartilage matrix components (perlecan, DMP1, and MEPE) in developing primary (tibial) and secondary (condylar) cartilage. Perlecan mRNA expression was first detected in newly formed chondrocytes in tibial cartilage at E13.0, but this expression decreased in hypertrophic chondrocytes at E14.0. In contrast, at E15.0, perlecan mRNA was first detected in the newly formed chondrocytes of condylar cartilage; these chondrocytes had characteristics of hypertrophic chondrocytes, which confirmed the previous observation that progenitor cells of developing secondary cartilage rapidly differentiate into hypertrophic chondrocytes. DMP1 mRNA was detected in many chondrocytes within the lower hypertrophic cell zone in tibial cartilage at E14.0. In contrast, DMP1 mRNA expression was only transiently detected in a few chondrocytes of condylar cartilage at E15.0. Thus, DMP1 may be less important in the developing condylar cartilage than in the tibial cartilage. Another purpose of this study was to test the hypothesis that MEPE may be a useful marker molecule for cartilage. MEPE mRNA was not detected in any chondrocytes in either tibial or condylar cartilage; however, MEPE immunoreactivity was detected throughout the cartilage matrix. Western immunoblot analysis demonstrated that MEPE antibody recognized two bands, one of 67 kDa and another of 59 kDa, in cartilage-derived samples. Thus MEPE protein may gradually accumulate in the cartilage, even though mRNA expression levels were below the limits of detection of in situ hybridization. Ultimately, we could not designate MEPE as a marker molecule for cartilage, and would modify our original hypothesis.

An Immunohistochemical Study of Matrix Components in Early-Stage Vascular Canals Within Mandibular Condylar Cartilage in Midterm Human Fetuses.

Matrix components of vascular canals (VCs) in human fetal mandibular condylar cartilage (15-16 weeks of gestation) were analyzed by immunohistochemistry. Prevascular canals (PVCs), consisting of spindle-shaped cells without capillary invasion, were observed within the cartilage. Intense immunoreactivity for collagen type I, weak immunoreactivity for aggrecan and tenascin-C, weak hyaluronan (HA) staining, and abundant argyrophilic fibers in PVCs indicated that they contain noncartilaginous fibrous connective tissues that was different from those in the perichondrium/periosteum. These structural and immunohistochemical features of PVCs are different from those of previously reported cartilage canals of the long bone. Capillaries entered the VCs from the periosteum and ascended through VCs. Following capillary invasion, loose connective tissue had formed in the lower part of VCs, and immunoreactivity for collagen types I and III, tenascin-C, and HA staining was evident in the matrix of loose connective tissue. No chondroclasts or osteogenic cells were seen at the front of capillary invasion, although small, mononuclear tartrate-resistant acid phosphatase (TRAP)-positive cells were present. Meanwhile, TRAP-positive, multinucleated chondroclasts and flattened, osteoblast-like cells were observed in the loose connective tissue at the lower part of VCs. These results may indicate slow progress of endochondral ossification in human fetal mandibular condyle. Further, unique matrix components in PVCs/VCs, which were different from those in cartilage canals in long bone, may reflect the difference of speed of endochondral ossification in cartilage canals and human fetal mandibular condyles.

MicroRNA-200a Regulates the Development of Mandibular Condylar Cartilage.

Mandibular condylar cartilage (MCC) is classified as secondary cartilage, the histologic structure of which is unique from that of primary cartilage. MicroRNA (miRNA) is a small noncoding RNA that binds to the messenger RNA (mRNA) target to repress its translation and plays an important role in cell differentiation, proliferation, and death. Microarray analysis revealed that miR-200a was characteristically expressed during embryonic development. We hypothesized that miR-200a may be involved in regulating the formation of cartilage during MCC growth. We investigated the function of miR-200a by transfecting an inhibitor or mimic into MCC organ and cell cultures. A histologic examination revealed the localized inhibitory effects of the miR-200a mimic and widespread enhancing effects of the inhibitor on chondrocytic differentiation in the MCC organ culture system. An immunohistochemical examination and gene expression analysis demonstrated that the miR-200a inhibitor enhanced chondrogenesis, while the mimic had the opposite effect by enhancing cell proliferation. Quantitative reverse transcription polymerase chain reaction analysis revealed that miR-200a downregulated the gene expression of chondrocyte markers. Moreover, transfection of the miR-200a mimic into ATDC5 cells repressed the formation of the cartilaginous matrix. These results indicate that miR-200a contributed to chondrogenesis in developing MCC by controlling proliferation and differentiation in MCC cells.

Fetal jaw movement affects Ihh signaling in mandibular condylar cartilage development: the possible role of Ihh as mechanotransduction mediator.

OBJECTIVE: Jaw movement is an important mechanical factor for prenatal development of the condylar cartilage of mandible. Fetal jaw movement restriction has been shown to cause deformity of the mandibular condyle. We hypothesized that this treatment affects the expression of mechanosensitive molecules, namely Indian hedgehog (Ihh) and Parathyroid hormone related protein (PTHrP) in the condyle. EXPERIMENTAL METHODS: We restrained jaw movement by suturing the jaw of E15.5 mouse embryos and allowed them to develop until E18.5 using exo utero system, and analyzed them by immunohistochemistry and in situ hybridization methods. RESULTS: Morphological, histomorphometric and immunohistochemical study showed that the mandibular condylar cartilage was reduced and deformed, the volume and total cell numbers in the condylar cartilage were also reduced, and number and/or distribution of 5-bromo-2'-deoxyuridine-positive cells, Ihh-positive cells in the mesenchymal and pre-hypertrophic zones were significantly and correspondingly decreased in the sutured group. Using in situ hybridization, reduced expression of Ihh, PTHrP and their related receptors were observed in condylar cartilage of the sutured embryos. CONCLUSIONS: Our results revealed that the altered mechanical stress induced by prenatal jaw movement restriction decreased proliferating cells, the amount of cartilage, and altered expression of the Ihh and PTHrP, suggesting that Ihh act as mechanotransduction mediators in the development of mandibular condylar cartilage.

Distribution of matrix proteins in perichondrium and periosteum during the incorporation of Meckel's cartilage into ossifying mandible in midterm human fetuses: an immunohistochemical study.

Immunohistochemical localization of versican and tenascin-C were performed; the periosteum of ossifying mandible and the perichondrium of Meckel's cartilage, of vertebral cartilage, and of mandibular condylar cartilage were examined in midterm human fetuses. Versican immunoreactivity was restricted and evident only in perichondrium of Meckel's cartilage and vertebral cartilage; conversely, tenascin-C immunoreactivity was only evident in periosteum. Therefore, versican and tenascin-C can be used as molecular markers for human fetal perichondrium and fetal periosteum, respectively. Meckel's cartilage underwent endochondral ossification when it was incorporated into the ossifying mandible at the deciduous lateral incisor region. Versican immunoreactivity in the perichondrium gradually became weak toward the anterior primary bone marrow. Tenascin-C immunoreactivity in the primary bone marrow was also weak, but tenascin-C positive areas did not overlap with versican-positive areas; therefore, degradation of the perichondrium probably progressed slowly. Meanwhile, versican-positive perichondrium and tenascin-C-positive periosteum around the bone collar in vertebral cartilage were clearly discriminated. Therefore, the degradation of Meckel's cartilage perichondrium during endochondral ossification occurred at a different rate than did degradation of vertebral cartilage perichondrium. Additionally, the perichondrium of mandibular condylar cartilage showed tenascin-C immunoreactivity, but not versican immunoreactivity. That perichondrium of mandibular condylar cartilage has immunoreactivity characteristic of other periosteum tissues may indicate that this cartilage is actually distinct from primary cartilage and representative of secondary cartilage.

Morphometric growth relationships of the immature human mandible and tongue.

The masticatory apparatus is a highly adaptive musculoskeletal complex comprising several relatively independent structural components, which assist in functions including feeding and breathing. We hypothesized that the tongue is elemental in the maintenance of normal ontogeny of the mandible and in its post-natal growth and development, and tested this using a morphometric approach. We assessed tongue and mandibular measurements in 174 (97 male) human cadavers. Landmark lingual and mandibular data were gathered individuals aged between 20 gestational weeks and 3 yr postnatal. In this analysis, geometric morphometrics assisted in visualizing the morphometrical growth changes in the mandible and tongue. A linear correlation in conjunction with principal component analysis further visualized the growth relationship between these structures. We found that the growth of the tongue and mandible were intrinsically linked in size and shape between 20 gestational weeks and 24 months postnatal. However, the mandible continued to change in shape and size into the 3rd yr of life, whereas the tongue only increased in size over this same period of time. These findings provide valuable insights into the allometric growth relationship between these structures, potentially assisting the clinician in predicting the behaviour of these structures in the assessment of malocclusions.

Double-stranded RNA-dependent protein kinase regulates insulin-stimulated chondrogenesis in mouse clonal chondrogenic cells, ATDC-5.

Double-stranded RNA-dependent protein kinase (PKR) is an interferon-induced protein that has been identified and characterized as a translational inhibitor in an interferon-regulated antiviral pathway. PKR is also reported to play important roles in the regulation of cell growth and differentiation. We have previously demonstrated that PKR inactivation suppresses osteoblast calcification and osteoclast formation. However, reports concerning the roles of PKR in chondrogenesis are limited. In this study, we have demonstrated that PKR is required for the in vitro differentiation of the mouse clonal chondrogenic cell line ATDC-5. ATDC-5 cells treated with insulin differentiated into chondrocytes and produced an alcian-blue-positive cartilage matrix. The protein expression of signal transducers and activators of transcription (STAT) peaked at day 7 of differentiation, whereas the expression of SRY-box-containing gene 9 (Sox-9), which is a transcription factor for chondrocyte differentiation, increased gradually. When the cells were treated with a PKR inhibitor (2-aminopurine), the cartilage matrix formation decreased. The protein expression of STAT1 continued to increase up to day 21, whereas the expression of Sox-9 was low and did not increase. We also demonstrated that PKR was localized to a marginal region of the mandibular condyle cartilage in mouse embryos. Our findings suggest that PKR has important functions in the differentiation of chondrocytes through the modulation of STAT1 and Sox-9 expression.

An in situ hybridization study of the insulin-like growth factor system in developing condylar cartilage of the fetal mouse mandible.

The objective of this study was to investigate the involvement of the insulin-like growth factor (IGF) system in the developing mandibular condylar cartilage and temporomandibular joint (TMJ). Fetal mice at embryonic day (E) 13.0-18.5 were used for in situ hybridization studies using [35S]-labeled RNA probes for IGF-I, IGF-II, IGF-I receptor (-IR), and IGF binding proteins (-BPs). At E13.0, IGF-I and IGF-II mRNA were expressed in the mesenchyme around the mandibular bone, but IGF-IR mRNA was not expressed within the bone. At E14.0, IGF-I and IGF-II mRNA were expressed in the outer layer of the condylar anlage, and IGF-IR mRNA was first detected within the condylar anlage, suggesting that the presence of IGF-IR mRNA in an IGF-rich environment triggers the initial formation of the condylar cartilage. IGFBP-4 mRNA was expressed in the anlagen of the articular disc and lower joint cavity from E15.0 to 18.5. When the upper joint cavity was formed at E18.5, IGFBP-4 mRNA expression was reduced in the fibrous mesenchymal tissue facing the upper joint cavity. Enhanced IGFBP-2 mRNA expression was first recognized in the anlagen of both the articular disc and lower joint cavity at E16.0 and continued expression in these tissues as well as in the fibrous mesenchymal tissue facing the upper joint cavity was observed at E18.5. IGFBP-5 mRNA was continuously expressed in the outer layer of the perichondrium/fibrous cell layer in the developing mandibular condyle. These findings suggest that the IGF system is involved in the formation of the condylar cartilage as well as in the TMJ.

Spry1 and spry2 are essential for development of the temporomandibular joint.

The temporomandibular joint (TMJ) is a specialized synovial joint essential for the function of the mammalian jaw. The main components of the TMJ are the mandibular condyle, the glenoid fossa of the temporal bone, and a fibrocartilagenous disc interposed between them. The genetic program for the development of the TMJ remains poorly understood. Here we show the crucial role of sprouty (Spry) genes in TMJ development. Sprouty genes encode intracellular inhibitors of receptor tyrosine kinase (RTK) signaling pathways, including those triggered by fibroblast growth factors (Fgfs). Using in situ hybridization, we show that Spry1 and Spry2 are highly expressed in muscles attached to the TMJ, including the lateral pterygoid and temporalis muscles. The combined inactivation of Spry1 and Spry2 results in overgrowth of these muscles, which disrupts normal development of the glenoid fossa. Remarkably, condyle and disc formation are not affected in these mutants, demonstrating that the glenoid fossa is not required for development of these structures. Our findings demonstrate the importance of regulated RTK signaling during TMJ development and suggest multiple skeletal origins for the fossa. Notably, our work provides the evidence that the TMJ condyle and disc develop independently of the mandibular fossa.

An immunohistochemical study on the localization of type II collagen in the developing mouse mandibular condyle.

The present chronological investigation assessed the distribution of type II collagen expression in the developing mouse mandibular condyle using immunohistochemical staining with respect to the anatomy of the anlage of the mandibular condyle, the histological characteristics of which were disclosed in our previous investigation. We analyzed fetuses, obtained by cross breeding of ICR strain mice, between 14.0 and 19.0 days post-conception (dpc) and pups on 1, 3, and 5 days post-natal (dpn) using immunohistochemical staining with 2 anti-type II collagen antibodies. The expression of type II collagen was first detected at 15.0 dpc in the lower part of the hypertrophic chondrocyte zone; thereafter, this type II collagen-positive layer was expanded and intensified (P1 layer). At 17.0 dpc, we identified a type II collagen-negative layer (N layer) around the P1 layer and we also identified another newly formed type II collagen-positive layer (P, layer) on the outer surface of the N layer. The most typical and conspicuous 3-layered distribution was observed at 1 dpn; thereafter, there was a reduction in the intensity of expression, and with it, the demarcation between the layers was weakened by 5 dpn. The P1 layer was derived from the central region of the core cell aggregate of the anlage of the mandibular condyle and participated in endochondral bone formation. The N layer was derived from the fringe of the core cell aggregate of the anlage, formed the bone collar at the side of the condyle by intramembranous bone formation, and showed a high level of proliferative activity at the vault. The P2 layer was formed from the outgrowth of the N layer, and could be considered as the secondary cartilage. The intensive expression of type II collagen from 17.0 dpc to 3 dpn was detected in the fibrous sheath covering the condylar head, which is derived from the peripheral cell aggregate of the anlage. Since its expression in the fibrous sheath was not detected in the neighboring section in the absence of hyaluronidase digestion, some changes in the extracellular matrix of the fibrous sheath appear to participate in the generation of the lower joint space. The results of the present investigation indicate that further studies are required to fully characterize the development of the mouse mandibular condyle.

Tissue interaction is required for glenoid fossa development during temporomandibular joint formation.

The mammalian temporomandibular joint (TMJ) develops from two distinct mesenchymal condensations that grow toward each other and ossify through different mechanisms, with the glenoid fossa undergoing intramembranous ossification while the condyle being endochondral in origin. In this study, we used various genetically modified mouse models to investigate tissue interaction between the condyle and glenoid fossa during TMJ formation in mice. We report that either absence or dislocation of the condyle results in an arrested glenoid fossa development. In both cases, glenoid fossa development was initiated, but failed to sustain, and became regressed subsequently. However, condyle development appears to be independent upon the presence of the forming glenoid fossa. In addition, we show that substitution of condyle by Meckel's cartilage is able to sustain glenoid fossa development. These observations suggest that proper signals from the developing condyle or Meckel's cartilage are required to sustain the glenoid fossa development.

Morphologic features of the fetal mandibular condyle: layers, canals and microvascular pattern.

During organogenesis the mandibular condyle is divided by a fibrovascular septum, the persistence of which in the growing cartilage can lead to a bifid condyle. In this study we have evaluated the morphology of 3rd trimester human fetal temporomandibular (TMJ) specimens in order to determine the pattern of the vascular morphology associated with the layers and vascular canals (VCs) of the developing condyle (covering layers and condyle proper). Eleven human fetuses of 27-38cm crown-rump length were used for histological (hematoxylin-eosin, Van Gieson stain) and immunohistochemical evaluation (antibodies for bcl2 and CD34) and another two of 24 and 31cm, for TMJ microvasculature studies after black ink injections. With increasing fetal age, the intermediate loose lamina (LL) of the condylar proliferative layer evolves from a vascular-mesenchymal to a fibrillar pattern, via a transitory stage of a clear space that may be misdiagnosed as lower joint cavity (LJC). Within the condyle proper VCs may be present on its entire sagittal length, deepening variably towards the erosive zone and opened superiorly in the LL loose layer. Vessels of the evolving LL enter the condyle, directly or through the VCs; these vessels retract peripherally with increasing age and the intrinsic vessels of the condyle supplied from the erosive zone become prevalent. Vascular morphogenesis at the level of the LL seems comparable to that at the level of the LJC where characteristic glomeruli regress with increasing age. Lack of vascular regression and closure of central V-shaped defects of the condyle, as observed in 2/22 condyles, may represent a developmental substrate for condylar bifidism or a predisposing condition weakening the condyle, and making it more sensitive to trauma in childhood.

Morphological observation of process of mouse temporomandibular joint formation.

The aim of this study was to clarify the developmental mechanism of the temporomandibular joint (TMJ) cavity, using the relationship between Meckel's cartilage and the mandible to morphologically observe the process of TMJ formation in mouse fetuses. We investigated the involvement of apoptosis in the development of the mouse TMJ cavity. We attempted to 3-dimensionally clarify the developmental process of the mandible and Meckel's cartilage by observing the developmental process optically and reconstructing 3-dimensional images to observe 3-dimensional locations of the mandible and Meckel's cartilage. Formation of the upper joint cavity began on embryonal day 16, and a complete joint cavity was formed on embryonal day 18. Formation of the lower joint cavity began on embryonal day 18, and formation was almost completed on embryonal day 19. Meckel's cartilage adjacent to the mandible decreased with development of the mandible but was vestigial on embryonal day 19. The posterior region of Meckel's cartilage developed toward the posterior direction, and it was 3-dimensionally confirmed that the mandible and Meckel's cartilage were separated. Histological observation by the TUNEL method revealed the presence of solitary and diffuse apoptotic cells not only in the joint cavity, but also around the condyle.

Sulfotransferase Ndst1 is needed for mandibular and TMJ development.

Heparan sulfate proteoglycans (HS-PGs) regulate several developmental processes, but their possible roles in mandibular and TMJ formation are largely unclear. To uncover such roles, we generated mice lacking Golgi-associated N-sulfotransferase 1 (Ndst1) that catalyzes sulfation of HS-PG glycosaminoglycan chains. Ndst1-null mouse embryos exhibited different degrees of phenotypic penetrance. Severely affected mutants lacked the temporomandibular joint and condyle, but had a mandibular remnant that displayed abnormal tooth germs, substandard angiogenesis, and enhanced apoptosis. In mildly affected mutants, the condylar growth plate was dysfunctional and exhibited thicker superficial and polymorphic cell zones, a much wider distribution of Indian hedgehog signaling activity, and ectopic ossification along its lateral border. Interestingly, mildly affected mutants also exhibited facial asymmetry resembling that seen in individuals with hemifacial microsomia. Our findings indicate that Ndst1-dependent HS sulfation is critical for mandibular and TMJ development and allows HS-PGs to exert their roles via regulation of Ihh signaling topography and action.

The immunolocalization and possible role of c-Met (MET, hepatic growth factor receptor) in the developing human fetal mandibular condyle.

The c-Met system is involved in skeletogenesis and is expressed in the cartilage of growth plates. However, the localization and role of c-Met during endochondral ossification of developing mandibular condyles or during intramembranous ossification has not yet been elucidated. In this study, c-Met was examined immunohistochemically in the mandibles of human fetuses during weeks 9 and 16 of pregnancy. c-Met was immunolocalised in the whole area of the developing mandible, although to different extents. In the intramembranous bone, mesenchymal cells showed a weak immunopositivity. Osteoprogenitor cells demonstrated a moderate immunopositivity for c-Met, while osteoblasts and osteocytes showed a very strong immunolabelling of c-Met. In the developing mandibular condyles, c-Met immunopositivity increased gradually throughout the proliferative layer towards the pre-hypertrophic cell layer, whereas the cells of the hypertrophic layer were weakly immunopositive. These findings have demonstrated, for the first time, the prominent immunolocalization of c-Met in osteogenic and chondrogenic tissues of developing human mandibles, which indicates possible functions for this receptor during mandibular development.