Shiho Honma

Osterix Regulates Calcification and Degradation of Chondrogenic Matrices through Matrix Metalloproteinase 13 (MMP13) Expression in Association with Transcription Factor Runx2 during Endochondral Ossification

Background: Molecular mechanisms controlling the late stages of endochondral ossification are unclear. Results: Osterix functions as a downstream and transcriptional partner of Runx2 and induces MMP13 during chondrocyte differentiation. Conclusion: Osterix is essential for late-stage endochondral ossification. Significance: Osterix affects the ossification of cartilage matrices and matrix vesicles and might be involved in the development of osteoarthritis and related disorders. Endochondral ossification is temporally and spatially regulated by several critical transcription factors, including Sox9, Runx2, and Runx3. Although the molecular mechanisms that control the late stages of endochondral ossification (e.g. calcification) are physiologically and pathologically important, these precise regulatory mechanisms remain unclear. Here, we demonstrate that Osterix is an essential transcription factor for endochondral ossification that functions downstream of Runx2. The global and conditional Osterix-deficient mice studied here exhibited a defect of cartilage-matrix ossification and matrix vesicle formation. Importantly, Osterix deficiencies caused the arrest of endochondral ossification at the hypertrophic stage. Microarray analysis revealed that matrix metallopeptidase 13 (MMP13) is an important target of Osterix. We also showed that there exists a physical interaction between Osterix and Runx2 and that these proteins function cooperatively to induce MMP13 during chondrocyte differentiation. Most interestingly, the introduction of MMP13 stimulated the calcification of matrices in Osterix-deficient mouse limb bud cells. Our results demonstrated that Osterix was essential to endochondral ossification and revealed that the physical and functional interaction between Osterix and Runx2 were necessary for the induction of MMP13 during endochondral ossification.

DISTRIBUTION PATTERN OF INHIBITORY AND EXCITATORY SYNAPSES IN THE DENDRITIC TREE OF SINGLE MASSETER ALPHA -MOTONEURONS IN THE CAT

Little is known about the differences in the distributions of inhibitory and excitatory synapses in the dendritic tree of single motoneurons in the brainstem and spinal cord. In this study, the distribution of γ‐aminobutyric acid (GABA)‐, glycine‐, and glutamate‐like immunoreactivity in axon terminals on dendrites of cat masseter α‐motoneurons, stained intracellularly with horseradish peroxidase, was examined by using postembedding immunogold histochemistry in serial ultrathin sections. The dendritic tree was divided into three segments: primary (Pd) and distal (Dd) dendrites and intermediate (Id) dendrites between the two segments. Quantitative analysis of 175, 279, and 105 boutons synapsing on 13 Pd, 54 Id, and 81 Dd, respectively, was performed. Fifty percent of the total number of studied boutons were immunopositive for GABA and/or glycine and 48% for glutamate. Among the former, 27% showed glycine immunoreactivity only and 14% were immunoreactive to both glycine and GABA. The remainder (9%) showed immunoreactivity for GABA only. As few as 3% of the boutons were immunonegative for the three amino acids. Most boutons immunoreactive to inhibitory amino acid(s) contained a mixture of spherical, oval, and flattened synaptic vesicles. Most boutons immunoreactive to excitatory amino acid contained clear, spherical, synaptic vesicles with a few dense‐cored vesicles. When comparisons of the inhibitory and excitatory boutons were made between the three dendritic segments, the proportion of the inhibitory to the excitatory boutons was high in the Pd (60% vs. 37%) but somewhat low in the Id (46% vs. 52%) and Dd (44% vs. 53%). The percentage of synaptic covering and packing density of the inhibitory synaptic boutons decreased in the order Pd, Id, and Dd, but this trend was not applicable to the excitatory boutons. The present study provides possible evidence that the spatial distribution patterns of inhibitory and excitatory synapses are different in the dendritic tree of jaw‐closing α‐motoneurons. J. Comp. Neurol. 414:454–468, 1999.

Quantitative ultrastructure of physiologically identified premotoneuron terminals in the trigeminal motor nucleus in the cat

Little is known about the ultrastructure of synaptic boutons contacting trigeminal motoneurons. To address this issue, physiologically identified premotor neurons (n = 5) in the rostrodorsomedial part of the oral nucleus (Vo.r) were labeled by intracellular injections of horseradish peroxidase (HRP) in cats. The ultrastructure of 182 serially sectioned axon terminals from the five neurons was both qualitatively and quantitatively analyzed. In addition, the effects of the glycine antagonist strychnine, GABAA antagonist bicuculline, NMDA antagonist 2‐amino‐5‐phosphonovalerate (APV), and non‐NMDA antagonist 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX) on Vo.r‐induced postsynaptic potentials in trigeminal motoneurons (n = 11) were examined to evaluate potential signaling substances of the premotor neurons. Labeled boutons made synaptic contacts with either jaw‐closing or ‐opening motoneurons. All the boutons contained pleomorphic vesicles, and most formed a single symmetric synapse either on the somata or on primary dendrites. Morphometric analyses indicated that bouton volume, bouton surface area, apposed surface area, total active zone area, and mitochondrial volume were not different between boutons on jaw‐closing and ‐opening motoneurons. Vesicle number and density, however, were higher for boutons on jaw‐closing motoneurons. The five morphological parameters were positively correlated with bouton volume. Vesicle density was the exception, which tending to be negatively correlated. Intravenous infusion of strychnine or bicuculline suppressed Vo.r‐induced inhibitory postsynaptic potentials (IPSPs) in jaw‐closing motoneurons. Abolition of Vo.r‐induced excitatory postsynaptic potentials in jaw‐opening motoneurons with APV and CNQX unmasked IPSPs. The present results suggest that premotor neurons in the Vo.r are inhibitory and that positive correlations between the ultrastructural parameters associated with synaptic release and bouton size are applicable to the interneurons, as they are in primary afferents. J. Comp. Neurol. 426:13–30, 2000.

Physiologic and morphologic properties of motoneurons and spindle afferents innervating the temporal muscle in the cat.

Little is known about physiology and morphology of motoneurons and spindle afferents innervating the temporalis and on synaptic connections made between the two. The present study was aimed at investigating the above issues at the light microscopic level by using the intracellular recording and horseradish peroxidase or biotinamide labeling techniques and by the use of succinylcholine (SCh) for the classification of spindle afferents in the cat. Temporalis motoneurons had dendritic trees that ranged from a spherical form to an egg‐shaped form. The shape deformation was more prominent for the dendritic trees made by motoneurons located closer to the nuclear border. No axon collaterals of the motoneurons were detected. On the basis of the values for the dynamic index after SCh infusion, temporalis spindle afferents were classified into two populations: presumptive groups Ia and II. The spindle afferents terminated mainly in the supratrigeminal nucleus (Vsup), region h, and the dorsolateral subdivision (Vmo.dl) of the trigeminal motor nucleus (Vmo). The proportion of group Ia afferent terminals was lower in the Vsup than that of group II afferents. In the Vmo.dl, the proportion of group Ia afferent terminals was nearly even throughout the nucleus, but that of group II afferent terminals increased in the more outlying regions. The proportion of terminal distribution in the central region of Vmo.dl was higher for group Ia than group II. The frequency of contacts (presumptive synapses) made by a single spindle afferent on a motoneuron was higher for group Ia than group II. The present study provided evidence that the central organization of spindle afferent neurons is different between groups Ia and II. J. Comp. Neurol. 406:29–50, 1999.

Quantitative analysis of the dendritic architectures of cat hypoglossal motoneurons stained intracellularly with horseradish peroxidase.

Little is known about the dendritic architecture of cat hypoglossal motoneurons. Thus, the present study was done to provide quantitative descriptions of hypoglossal motoneurons and to determine correlations between dendritic size parameters by using the intracellular horseradish peroxidase (HRP) injection technique in the cat. Twelve hypoglossal motoneurons stained with HRP were antidromically activated by stimulation applied to the medial branch of hypoglossal nerve. Eight (type I) and four (type II) of the 12 motoneurons were located in the ventral and dorsal parts of the ventromedial subnucleus of hypoglossal nucleus, respectively. The somatodendritic morphology of the two types of neurons was remarkably different, especially in the dendritic arborization pattern. The type I neurons established an egg‐shaped dendritic tree that was distributed through the nucleus to the reticular formation; the type II dendritic tree was confined within the nucleus and presented a rostrocaudally oriented, mirror‐image, fan‐shape appearance. The total dendritic area and length and the number of terminations and branch points were significantly larger for type I than for type II neurons. For the two types of neuron, there was a positive correlation between stem dendritic diameter and several dendritic size parameters. Although the slopes of the regression lines were slightly different between the two, these were not statistically significant. The present study provides evidence that hypoglossal motoneurons located in the ventromedial subnucleus could be divided into two types according to the dendritic arborization pattern and quantitative analysis of the dendritic tree and according to neuronal location and suggests that the two types of hypoglossal motoneurons can be viewed as intrinsically distinct cell types: type I and type II, which innervate extrinsic and intrinsic muscles, respectively. In addition, the morphometric analysis made it possible to estimate the size of the dendritic tree by measuring the stem dendritic diameter. J. Comp. Neurol. 405:345–358, 1999.

The development of terminal Schwann cells associated with periodontal Ruffini endings in the rat incisor ligament.

The postnatal development of the terminal Schwann cell, an analogue of the lamellar cell in cutaneous sensory receptors, was examined by histochemistry for non-specific cholinesterase and immunohistochemistry for S-100 protein in the periodontal Ruffini endings of the rat incisor. Double immunohistochemistry for S-100 protein and protein gene product 9.5 (PGP 9.5) was also performed to examine the relationship between terminal Schwann cells and axons. Histochemistry for non-specific cholinesterase was able to demonstrate the age-related development of the terminal Schwann cells; the morphology and distribution of the developing terminal Schwann cells became almost identical to those in adults during postnatal days 15-18. Axons showing PGP 9.5-like immunoreactivity elongated and expanded after arrangement of terminal Schwann cells in the alveolus-related part. This suggests that the terminal Schwann cell is important in the development and maturation of the periodontal Ruffini endings.

Morphological differences between fast and slowly adapting lingual afferent terminations in the principal and oral nuclei in the cat

Previous studies indicated that fast‐adapting (FA) and slowly adapting (SA) mechanoreceptive afferents innervating the facial or intraoral structures give rise to morphologically distinct terminal arbors in the individual subdivisions of the trigeminal sensory nuclear complex. The present study examined the collateral morphologies of lingual afferents in the nuclei principalis (Vp) and oralis (Vo) of the cat. Seven FA and six SA lingual afferents were physiologically characterized and stained by the intra‐axonal horseradish peroxidase (HRP) injection technique. The two types of afferents established terminal arbors in the dorsomedial subdivision (Vpd) of the Vp, and the rostrodorsomedial (Vo.r) and dorsomedial subdivisions (Vo.dm) of the Vo, but the collateral morphologies are different between the two types. The FA afferents gave rise to mediolaterally extended oblong arbors in each subdivision, but the arbors were better developed in the Vo.r than in the Vpd and Vo.dm. The number of collaterals, intercollateral distance, number of boutons per collateral, and bouton size were also different among the subdivisions. The SA afferents were divided into two subtypes; one had a preferential projection into the Vpd or the Vo.r and Vo.dm, and others lacked a selected projection. Although the shape of their arbors varied from a stringy form to a roundish form, the general profile was denser, better developed, and rounder than that of FA afferents in each subdivision. The intercollateral distance and bouton size were different among the subdivisions. The number of boutons per collateral, bouton density, and bouton size were larger in SA than FA afferents in each subdivision. The present study demonstrated that two functionally distinct lingual afferents manifest unique morphological differences in the Vpd and Vo. J. Comp. Neurol. 396:64–83, 1998.

Trps1 is necessary for normal temporomandibular joint development

Mutation of the human TRPS1 gene leads to trichorhinophalangeal syndrome (TRPS), which is characterized by an abnormal development of various organs including the craniofacial skeleton. Trps1 has recently been shown to be expressed in the jaw joints of zebrafish; however, whether Trps1 is expressed in the mammalian temporomandibular joint (TMJ), or whether it is necessary for TMJ development is unknown. We have analyzed (1) the expression pattern of Trps1 during TMJ development in mice and (2) TMJ development in Trps1 knockout animals. Trps1 is expressed in the maxillo-mandibular junction at embryonic day (E) 11.5. At E15.5, expression is restricted to the developing condylar cartilage and to the surrounding joint disc progenitor cells. In Trps1 knockout mice, the glenoid fossa of the temporal bone forms relatively normally but the condylar process is extremely small and the joint disc and cavities do not develop. The initiation of condyle formation is slightly delayed in the mutants at E14.5; however, at E18.5, the flattened chondrocyte layer is narrowed and most of the condylar chondrocytes exhibit precocious chondrocyte maturation. Expression of Runx2 and its target genes is expanded toward the condylar apex in the mutants. These observations underscore the indispensable role played by Trps1 in normal TMJ development in supporting the differentiation of disc and synoviocyte progenitor cells and in coordinating condylar chondrocyte differentiation.

Postnatal development of synaptic inputs to rat masseter motoneurons

We examined postnatal changes in rat masseter motoneuron morphology and the density of synaptic inputs to masseter motoneurons using retrograde labeling combined with synaptophysin immunohistochemistry. The cross-sectional area and perimeter of masseter motoneurons increased through P21 whereas synaptic input density increased throughout the time frame sampled. Data suggest that changes in masseter motoneuron morphology and the density of its synaptic input contribute to the maturation of mastication behavior.

Quantitative analysis of the dendritic architectures of single jaw-closing and jaw-opening motoneurons in cats.

Little is known about the dendritic architectures of trigeminal motoneurons innervating antagonistic muscles. Thus, the aim of the present study was to provide a quantitative description of jaw-closing (JC) and jaw-opening (JO) alpha motoneurons and to determine geometrical similarities and differences of the dendritic tree between the two. Seven JC alpha motoneurons and four JO alpha motoneurons were intracellulary labeled with horseradish peroxidase (HRP) in the cat and quantitatively analyzed with a computer-assisted three-dimensional system. The dendritic tree of JC alpha motoneurons was confined within the JC motor nucleus, despite locations of the cell body. In contrast, JO alpha motoneurons generated extensive extranuclear dendrites in the reticular formation. The branching pattern of proximal dendritic segments was simpler in the JC than in the JO alpha motoneurons. Despite these differences, the mean values of dendritic parameters examined per neuron were not different between the two kinds of alpha motoneurons, and the stem dendrite diameter was positively correlated with several dendritic parameters in a linear manner. The present study provides new evidence that underlying design principles of the geometry of the dendritic tree are not concerned with the differences in configuration and branching pattern of the dendritic tree of trigeminal alpha motoneurons innervating antagonistic muscles. In addition, we estimated the number of excitatory and inhibitory synapses covering dendrites of single JC alpha motoneurons.