Masahiko Taniguchi

Osteoprotection by semaphorin 3A

The bony skeleton is maintained by local factors that regulate bone-forming osteoblasts and bone-resorbing osteoclasts, in addition to hormonal activity. Osteoprotegerin protects bone by inhibiting osteoclastic bone resorption, but no factor has yet been identified as a local determinant of bone mass that regulates both osteoclasts and osteoblasts. Here we show that semaphorin 3A (Sema3A) exerts an osteoprotective effect by both suppressing osteoclastic bone resorption and increasing osteoblastic bone formation. The binding of Sema3A to neuropilin-1 (Nrp1) inhibited receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast differentiation by inhibiting the immunoreceptor tyrosine-based activation motif (ITAM) and RhoA signalling pathways. In addition, Sema3A and Nrp1 binding stimulated osteoblast and inhibited adipocyte differentiation through the canonical Wnt/β-catenin signalling pathway. The osteopenic phenotype in Sema3a−/− mice was recapitulated by mice in which the Sema3A-binding site of Nrp1 had been genetically disrupted. Intravenous Sema3A administration in mice increased bone volume and expedited bone regeneration. Thus, Sema3A is a promising new therapeutic agent in bone and joint diseases.

Semaphorins guide the entry of dendritic cells into the lymphatics by activating myosin II

The recirculation of leukocytes is essential for proper immune responses. However, the molecular mechanisms that regulate the entry of leukocytes into the lymphatics remain unclear. Here we show that plexin-A1, a principal receptor component for class III and class VI semaphorins, was crucially involved in the entry of dendritic cells (DCs) into the lymphatics. Additionally, we show that the semaphorin Sema3A, but not Sema6C or Sema6D, was required for DC transmigration and that Sema3A produced by the lymphatics promoted actomyosin contraction at the trailing edge of migrating DCs. Our findings not only demonstrate that semaphorin signals are involved in DC trafficking but also identify a previously unknown mechanism that induces actomyosin contraction as these cells pass through narrow gaps.

Increased proximal bifurcation of CA1 pyramidal apical dendrites in sema3A mutant mice

Semaphorin‐3A (Sema3A) is an attractive guidance molecule for cortical apical dendrites. To elucidate the role of Sema3A in hippocampal dendritic formation, we examined the Sema3A expression pattern in the perinatal hippocampal formation and analyzed hippocampal dendrites of the brains from young adult sema3A mutant mice. Sema3A protein was predominantly expressed in the hippocampal plate and the inner marginal zone at the initial period of apical dendritic growth. Neuropilin‐1 and plexin‐A, the receptor components for Sema3A, were also localized in the same regions. The Golgi impregnation method revealed that in wildtype mice more than 90% of hippocampal CA1 pyramidal neurons extended a single trunk or apical trunks bifurcated in stratum radiatum. Seven percent of the pyramidal neurons showed proximal bifurcation of apical trunks in stratum pyramidale or at the border of the stratum pyramidale and stratum radiatum. In sema3A mutant mice, proximally bifurcated apical dendrites were increased to 32%, while the single apical dendritic pyramidal neurons were decreased. We designate this phenotype in sema3A mutant mice as “proximal bifurcation.” In the dissociated culture system, approximately half of the hippocampal neurons from wildtype mice resembled pyramidal neurons, which possess a long, thick, and tapered dendrite. In contrast, only 30% of the neurons from sema3A mutants exhibited pyramidal‐like morphology. Proximal bifurcation of CA1 pyramidal neurons was also increased in the mutant mice of p35, an activator of cyclin‐dependent kinase 5 (Cdk5). Thus, Sema3A may facilitate the initial growth of CA1 apical dendrites via the activation of p35/Cdk5, which may in turn signal hippocampal development. J. Comp. Neurol. 516:360–375, 2009.

Brain Endothelial Cells Control Fertility through Ovarian-Steroid–Dependent Release of Semaphorin 3A

Endothelial-cell–derived Sema3A promotes axonal outgrowth and plasticity and thereby regulates neurohormone release in the adult rodent brain in response to the ovarian cycle.

Ca2+ induces macropinocytosis via F-actin depolymerization during growth cone collapse

Growth cone collapse occurs in repulsive axon guidance and is accompanied by a reduction in the surface area of the plasma membrane of growth cones. However, the mechanism of this reduction is unclear. Here, we show that during growth cone collapse, caffeine-induced Ca(2+) release from ryanodine-sensitive Ca(2+) stores triggers the formation of large vacuoles in growth cones by macropinocytosis, a clathrin-independent endocytosis for the massive retrieval of the cellular plasma membrane, and subsequent retrograde membrane transport. We observed a significant correlation of the area of caffeine-induced macropinosomes with growth cone collapse. We also detected macropinocytosis induced by semaphorin 3A, a typical repulsive cue, and correlation between the area of semaphorin 3A-induced macropinocytic vacuoles and growth cone collapse. Moreover, jasplakinolide, an inhibitor of F-actin depolymerization, blocked caffeine-induced macropinocytosis. We propose that the coordinated regulation of actin cytoskeletal reorganization and macropinocytosis-mediated retrograde membrane trafficking may contribute to Ca(2+)-induced axon growth inhibition.

Syntaxin 1B Suppresses Macropinocytosis and Semaphorin 3A-Induced Growth Cone Collapse

Growth cone collapse is a crucial process for repulsive axon guidance and is accompanied by a reduction in growth cone surface area. This process of reduction may be regulated by endocytosis; however, its molecular mechanism is unclear. Macropinocytosis is a clathrin-independent form of endocytosis in which large areas of plasma membrane can be engulfed. We have reported previously that macropinocytosis is induced in growth cones of chick dorsal root ganglion neurons by semaphorin 3A (Sema3A), a repulsive axon guidance cue, and that Sema3A-induced reduction in growth cone surface area and macropinocytic vacuole area were correlated, suggesting a positive role for macropinocytosis in Sema3A-induced growth cone collapse. In the present study, we found that syntaxin 1B (Syx1B), a membrane trafficking protein, is a negative regulator of macropinocytosis, and its expression is downregulated by Sema3A signaling. Macropinocytosis inhibitor ethylisopropylamiloride or Syx1B overexpression suppressed Sema3A-induced macropinocytosis and growth cone collapse. These results indicate that Syx1B couples macropinocytosis-mediated massive internalization of the plasma membrane to Sema3A-induced growth cone collapse.

Semaphorin 3A controls timing and patterning of the dental pulp innervation.

Timing and patterning of dental pulp innervation are strictly spatio-temporally regulated but it is still not known how they are controlled at molecular level. We analyzed postnatal innervation of the dental pulp in the mandibular first molar of mice deficient for Semaphorin 3A (Sema3A) axon repellant molecule. Immunohistochemical localization of nerve fibers on serial sections covering the whole tooth germs using anti-peripherin antibody revealed that nerve fibers were prematurely present within the preodontoblast layer next to the inner enamel epithelium already at PN0 in Sema3A(-/-) mice. In contrast, in the wild-type (Sema3A(+/+)) mice nerve fibers were seen in the pulp only after enamel formation at PN3. The nerves in Sema3A(-/-) pulp were notably defasciculated and thinner compared to that of Sema3A(+/+) mice. A premature formation of an abnormal, enlarged nerve plexus with a high number of arborizations was apparent in the pulp-dentin border target area in Sema3A(-/-) already at PN2 whereas in the wild-type mice the first sign of plexus formation was seen at PN7. The expression of mRNAs for Ngf, Gdnf and Ncam neuroregulatory molecules in mandibular molar as well as receptors for neurotrophic factors and class 3 semaphorins including Sema3A (TrkA, p75, TrkB, TrkC, Ret, Npn1, Npn2, PlxA4) in trigeminal ganglia were not altered in the Sema3A(-/-) mice. Collectively, this data show that Sema3A serves an essential role in molar tooth pulp innervation controlling the timing of nerve fiber penetration into the pulp, their patterning and the formation of nerve plexus at pulp-dentin border area, and provide further support for the hypothesis that tooth innervation is regulated by the coordinated activity of locally expressed neuroregulatory molecules exerting positive and negative influences on growing dental nerve fibers.

Development of the dorsal ramus of the spinal nerve in the mouse embryo: Involvement of semaphorin 3A in dorsal muscle innervation

The spinal nerve, which is composed of dorsal root ganglion (DRG) sensory axons and spinal motor axons, forms the dorsal ramus projecting to the dorsal musculature. By using the free‐floating immunohistochemistry method, we closely examined the spatiotemporal pattern of the formation of the dorsal ramus and the relationship between its projection to the myotome/dorsal musculature and semaphorin 3A (Sema3A), which is an axonal guidance molecule. In embryonic day (E) 10.5–E11.5 wild‐type mouse embryos, we clearly showed the existence of a waiting period for the dorsal ramus projection to the myotome. In contrast, in E10.5–E11.5 Sema3A‐deficient embryos, the dorsal ramus fibers projected beyond the edge of the myotome without exhibiting the waiting period for projection. These results strongly suggest that the delayed innervation by dorsal ramus fibers may be caused by Sema3A‐induced axon repulsion derived from the myotome. Next, by performing culture experiments, we confirmed that E12.5 mouse axons responded to Sema3A‐induced repulsion. Together, our results imply that Sema3A may play a key role in the proper development of the dorsal ramus projection.

Development of the dorsal ramus of the spinal nerve in the chick embryo: a close relationship between development and expression of guidance cues.

The spinal nerve, which is composed of dorsal root ganglion (DRG) axons and spinal motor axons, divides into ventral and dorsal rami. Although the development of the ventral ramus has been examined in considerable detail, that of the dorsal ramus has not. Therefore, we first examined the spatial-temporal pattern of the dorsal ramus formation in the chick embryo, with special reference to the projection to the dermamyotome and its derivatives. Next, we focused on two guidance molecules, chick semaphorin 3A (SEMA3A) and fibroblast growth factor 8 (FGF8), because these are the best candidates as molecules for controlling the dorsal ramus formation. Using in situ hybridization and immunohistochemistry methods, we clearly showed a close relationship between the spatial-temporal expression of SEMA3A/FGF8 and the projection of dorsal ramus fibers to the dorsal muscles. We further examined the axonal response of motor and DRG neurons to SEMA3A and FGF8. We showed that motor axons responded to both SEMA3A-induced repulsion and FGF8-induced attraction. On the other hand, DRG axons responded to SEMA3A-induced repulsion but not to FGF8-induced attraction. These findings suggest that FGF8-induced attraction may guide early motor axons beneath the myotome and that SEMA3A-induced repulsion may prevent these early motor axons from entering the myotome. Our results also imply that the loss of SEMA3A expression in the dorsal muscles may lead to the gross projection of the dorsal ramus fibers into the dorsal muscles. Together, SEMA3A and FGF8 may contribute to the proper formation of the dorsal ramus.

Fetal Laser Surgery Exo Utero in Mice

ABSTRACTu2002 It has been very difficult to investigate mammalian embryogenesis. Whole‐embryo culture systems allow the observation of mammalian embryogenesis and enable the manipulation of the embryos, but it is almost impossible to culture fetuses at the late gestational period. The exo utero method does allow the manipulation of mammalian fetuses at the late gestational period. In this review, the detailed method of exo ufero surgery in mice is described, and the advantages and disadvantages of the applications of laser equipment for fetal surgery are discussed.