Patterning of mammalian heterodont dentition within the upper and lower jaws.

Mammalian heterodont dentition is differentiated into incisors, canines, premolars, and molars in the mesial-distal direction, in both the upper and lower jaws. Although all the lower teeth are rooted in the mandible, the upper incisors are rooted in the premaxilla and the upper canine and the teeth behind it are in the maxilla. The present study uncovers ontogenetic backgrounds to these shared and differing mesiodistal patterns of the upper and lower dentition. To this end, we examined the dentition development of the house shrew, Suncus murinus, instead of the rodent model animals because the dentition of this primitive eutherian species includes all the tooth classes, and no toothless diastema region. In the shrew, the upper incisor-forming region extended over the medial nasal prominence and the mesial part of the maxillary prominence. Consequently, the maxillary and mandibular prominences were in a mirror-image relationship in terms of the mesiodistally differentiated tooth-forming regions and of the complementary gene expression pattern, with Bmp4 in the mesial and Fgf8 in the distal regions. This suggests shared molecular mechanisms regulating tooth class differentiation between the upper and lower jaws. However, the premaxillary bone appeared within the mesenchyme of the medial nasal prominence, but grew distally beyond the former epithelial boundary with the maxillary prominence to form, finally, the incisive (premaxillary-maxillary) suture just mesial to the canine. Therefore, the developmental locations of the upper incisors are not inconsistent with the classical osteological criterion of the upper canine by comparative odontologists.

A morphological analysis of thalamocortical axon fibers of rat posterior thalamic nuclei: a single neuron tracing study with viral vectors.

The rostral sector of the posterior thalamic nuclei (POm) is, together with the ventral posterior nuclei (VP), involved in somatosensory information processing in rodents. The POm receives inputs from the spinal cord and trigeminal nuclei and projects to the primary somatosensory (S1) cortex and other cortical areas. Although thalamocortical axons of single VP neurons are well known to innervate layer (L) 4 of the S1 cortex with distinct columnar organization, those of POm neurons have not been elucidated yet. In the present study, we investigated complete axonal and dendritic arborizations of single POm neurons in rats by visualizing the processes with Sindbis viruses expressing membrane-targeted fluorescent protein. When we divided the POm into anterior and posterior parts according to calbindin immunoreactivity, dendrites of posterior POm neurons were wider but less numerous than those of anterior neurons. More interestingly, axon fibers of anterior POm neurons were preferentially distributed in L5 of the S1 cortex, whereas those of posterior neurons were principally spread in L1 with wider and sparser arborization than those of anterior neurons. These results suggest that the POm is functionally segregated into anterior and posterior parts and that the 2 parts may play different roles in somatosensory information processing.

The morphological and histological characters of the male external genitalia of the house musk shrew, Suncus murinus.

External genitalia are the reproductive organs necessary for efficient copulation and internal fertilization in various mammalian species. Their morphogeneses display significant morphological and developmental differences among species. The house musk shrew, Suncus murinus (hereafter described as suncus) is a species of the order Insectivora, which has been considered as primitive and one of the earliest eutheria phylogenetically. Comparative anatomical analyses of phylogenetically different mammals will contribute to the better understanding of morphological diversity of external genitalia. This study performed various anatomical and histological analyses concerning the organization of the external genitalia of male suncus. It was shown that the external genitalia of suncus possessed a muscular structure, which we proposed as musculus ischiocavernosus dorsalis of suncus. The musculus ischiocavernosus dorsalis is originated from the inner surface of the tuber ischiadicum and was allocated adjacent to the corpus cavernosum penis. In addition, a pair of alpha-smooth muscle actin positive muscles was located bilaterally to the urethra. This unique morphology of the external genitalia of suncus males may provide a unique model system to investigate genital morphogenesis.

Correlative analysis of immunoreactivity in confocal laser-scanning microscopy and scanning electron microscopy with focused ion beam milling.

Recently, three-dimensional reconstruction of ultrastructure of the brain has been realized with minimal effort by using scanning electron microscopy (SEM) combined with focused ion beam (FIB) milling (FIB-SEM). Application of immunohistochemical staining in electron microscopy (EM) provides a great advantage in that molecules of interest are specifically localized in ultrastructures. Thus, we applied immunocytochemistry for FIB-SEM and correlated this immunoreactivity with that in confocal laser-scanning microcopy (CF-LSM). Dendrites of medium-sized spiny neurons in the rat neostriatum were visualized using a recombinant viral vector, which labeled the infected neurons with membrane-targeted GFP in a Golgi stain-like fashion. Moreover, the thalamostriatal afferent terminals were immunolabeled with Cy5 fluorescence for vesicular glutamate transporter 2 (VGluT2). After detection of the sites of terminals apposed to the dendrites by using CF-LSM, GFP and VGluT2 immunoreactivities were further developed for EM by using immunogold/silver enhancement and immunoperoxidase/diaminobenzidine (DAB) methods, respectively. In contrast-inverted FIB-SEM images, silver precipitations and DAB deposits were observed as fine dark grains and diffuse dense profiles, respectively, indicating that these immunoreactivities were as easily recognizable as those in the transmission electron microscopy (TEM) images. Furthermore, in the sites of interest, some appositions displayed synaptic specializations of an asymmetric type. Thus, the present method was useful in the three-dimensional analysis of immunocytochemically differentiated synaptic connections in the central neural circuit.

Development of deciduous and permanent dentitions in the upper jaw of the house shrew (Suncus murinus).

The diphyodont tooth replacement in mammals is characterized by a single replacement of a deciduous dentition by a permanent dentition. Despite its significance in mammalian biology and paleontology, little is known about the developmental mechanisms regulating the diphyodont replacement. Because the mouse never replaces its teeth, this study used the house shrew, Suncus murinus, as a model to investigate the control of the diphyodont replacement of a deciduous dentition by successions and additions of permanent teeth. Using morphological and gene expression analyses of serial sections, we have demonstrated the development of the upper dentition of the house shrew. In this species, the deciduous tooth germs are formed but soon become vestigial, whereas the successional and accessional (molar) germs are subsequently formed and developed. There are distinct Shh expression domains in the deciduous, successional, and accessional tooth germs, and those of the latter two germs are identified from the appearance of their primary enamel knots. The developmental sequence of tooth germs in the house shrew indicates that two adjacent primary enamel knots of the successional and accessional germs do not develop simultaneously, but with a constant time lag. We suggest that this mode of tooth succession and accession can be explained by a sequential inhibitory cascade model in which the timing of initiation and the spacing of tooth development are determined by the inhibition from the primary enamel knots of developmentally preceding adjacent tooth germs.

Effectable application of vascular endothelial growth factor to critical sized rat calvaria defects.

OBJECTIVE: An early vascular response for angiogenesis is essential for the normal progression of bone defect healing. Vascular endothelial growth factor (VEGF) is a potent inducer of angiogenesis. The aim of this study was to evaluate the effects of a poly (L,D-lactic-co-glycolic acid) (PLGA) membrane with VEGF encapsulated into PLGA microspheres on bone regeneration at bone defects in rat calvaria. STUDY DESIGN: Microspheres of PLGA incorporating VEGF(165) (VEGF microspheres) were prepared, and critical-size bone defects were created in rat calvaria. The VEGF microspheres, PLGA microspheres, or VEGF microspheres plus PLGA membrane were applied to the defects. Bone regeneration was evaluated using image analysis based on soft radiographic and histologic examination. RESULTS: Mature thick bone regeneration was observed in selected sites at bone defects that had been applied with VEGF microspheres/PLGA membrane compared with those that had been applied with the other treatments. CONCLUSION: A combination of VEGF microspheres and a PLGA membrane effectively enhances bone regeneration.

Development of heterodont dentition in house shrew (Suncus murinus).

Mammalian heterodont dentition comprises incisors, canines, premolars, and molars. Although there has been intensive research, the patterning of these specific tooth types has not yet been elucidated. In order for the gene expression data to be linked with tooth type determination, it is first necessary to determine precisely the incisor-, canine-, premolar-, and molar-forming regions in the jaw primordia. To accomplish this, we studied dentition development in the house shrew (Suncus murinus), which has retained all the tooth types, using three-dimensional reconstructions from serial histological sections and the Sonic hedgehog (Shh) expression patterns. Before the appearance of morphological signs of odontogenesis, Shh expression localized to the presumptive tooth-forming regions, in which the mesial and distal expression domains corresponded to the incisor- and premolar-forming regions, respectively. The upper incisor region was found to extend across the boundary between the frontonasal and the maxillary processes. The canine-forming regions later appeared in the intermediate portions of the maxillary and the mandibular processes. The molar-forming regions later appeared distal to the initially demarcated tooth-forming regions by secondary extension of the distal ends. The demarcation visualized by the Shh expression pattern in the jaw primordia of the house shrew probably represents the basic developmental pattern of mammalian heterodont dentition.

Expression of D1 but not D2 dopamine receptors in striatal neurons producing neurokinin B in rats.

Neostriatal projection neurons are known to be largely divided into two groups, striatoentopeduncular/striatonigral and striatopallidal neurons, which mainly express D1 and D2 dopamine receptors, respectively. Recently, a small population of neostriatal neurons have been reported to produce neurokinin B (NKB), and send their axons mainly to the basal forebrain regions. To reveal which type of dopamine receptors were expressed by these NKB-producing neurons, we examined rat striatal neurons by combining immunofluorescence labeling for preprotachykinin B (PPTB), the precursor of NKB, and fluorescence in situ hybridization labeling for dopamine receptors. Fluorescent signals for D1 receptor mRNA were detected in 85-89% of PPTB-immunopositive neurons in the neostriatum, accumbens nucleus and lateral stripe of the striatum, whereas almost no signal for D2 receptor was observed in PPTB-positive striatal neurons. To further reveal intracellular signaling downstream of D1 receptor in PPTB-producing neurons, we used a double immunofluorescence labeling method to study the localization of some substrates for protein kinase A (PKA), which was known to be activated by D1 receptor. Although only 3-7% of PPTB-immunopositive striatal neurons displayed immunoreactivity for dopamine- and cAMP-regulated phosphoprotein of 32 kDa, a well-known PKA substrate expressed in the two major groups of neostriatal projection neurons, 60-64% of PPTB-positive striatal neurons exhibited immunoreactivity for striatal-enriched tyrosine phosphatase. These results suggest that NKB-producing neostriatal neurons are similar to striatoentopeduncular/striatonigral neurons in the usage of dopamine receptor subtypes, but different from the two major groups of neostriatal projection neurons in terms of the downstream signaling of dopamine receptors.

Comparison of expression patterns of fibroblast growth factor 8, bone morphogenetic protein 4 and sonic hedgehog in jaw development of the house shrew, Suncus murinus.

To elucidate the mechanism underlying jaw development in mammals, we used a new laboratory animal, Suncus murinus (house shrew, an insectivore) as the subject for the investigation, because Suncus has all types of teeth (incisor, canine, premolar and molar) in its upper and lower jaws and is thought to be a good model animal having a general mammalian tooth pattern. At the start, by use of degenerate primers we cloned Suncus homologues of fibroblast growth factor 8 (sFgf8), bone morphogenetic protein 4 (sBmp4) and sonic hedgehog (sShh) genes from cDNA library derived from whole Suncus embryos at day 12 (E12). Thereafter, we examined the expression patterns of these genes in the jaw development of Suncus E11-16 embryos (for mouse E9.5-12 embryos). sFgf8 and sBmp4 were expressed in E11 but not in E15 and onward during orofacial development. sShh was expressed from E11 onward, and its expression was increased in the orofacial area. The expression pattern of sFgf8 in the maxillary and mandibular arches of E14 coincided with the area of the presumptive tooth arch. However, sShh and sBmp4 were expressed only in the outer area (= buccal/labial side) of presumptive tooth arch. Thus, these 3 genes showed specific expression pattern in jaw development of Suncus, and their distributions did not overlap each other except in a few regions. These findings suggest that sFgf8, sBmp4 and sShh have a specific function respectively during jaw development in Suncus murinus.