Tomoichiro Yamaai

Severe neuropathies in mice with targeted mutations in the ErbB3 receptor.

Neuregulins and their specific receptors, members of the ErbB family of tyrosine kinases, have been implicated in the control of growth and development of Schwann cells, specialized cells that wrap around nerve axons to provide electrical insulation. Here we use gene targeting to generate mice that lack ErbB3, a high-affinity neuregulin receptor. Homozygous erbB3 mutant embryos lack Schwann-cell precursors and Schwann cells that accompany peripheral axons of sensory and motor neurons. The initial development of motor neurons and sensory neurons of dorsal root ganglia occurs as it should, but at later stages most motor neurons (79%) and sensory neurons in dorsal root ganglia (82%) undergo cell death in erbB3 mutant embryos. Degeneration of the peripheral nervous system in erbB3 mutant pups is thus much more severe than the cell death in mice that lack neurotrophins or neurotrophin receptors,. We also show that ErbB3 functions in a cell-autonomous way during the development of Schwann cells, but not in the survival of sensory or motor neurons. Our results indicate that sensory and motor neurons require factors for their survival that are provided by developing Schwann cells.

Expression pattern of acidic and basic fibroblast growth factor genes in adult rat eyes

Although the retinal angiogenic and mitogenic factors have been identified to be acidic and basic fibroblast growth factors (aFGF and bFGF), little information has so far been available about the cells producing them and their function in retinal tissues. We found, by in situ hybridization, that the expression pattern of the aFGF gene differed remarkably from that of the bFGF gene in adult rat eyes. Our results demonstrated that the aFGF gene was produced by photoreceptor visual cells, neuronal cells in the inner nuclear layer and ganglion cells of the retina, in addition to pigment epithelial cells of the choroid, iris and ciliary body, and epithelial cells of the cornea, conjunctiva and lens, while bFGF was synthesized solely by the photoreceptor visual cells.

Expression of connective tissue growth factor/hypertrophic chondrocyte-specific gene product 24 (CTGF/Hcs24) during fracture healing

Localization and expression of connective tissue growth factor/hypertrophic chondrocyte-specific gene product 24 (CTGF/Hcs24) during fracture healing in mouse ribs were investigated. In situ hybridization demonstrated that CTGF/Hcs24 mRNA was remarkably expressed, especially in hypertrophic chondrocytes and proliferating chondrocytes, in the regions of regenerating cartilage on days 8 and 14 after fracture. CTGF/Hcs24 mRNA was also expressed in proliferating periosteal cells in the vicinity of the fracture sites on days 2 and 8, and in cells in fibrous tissue around the callus on day 8. Northern blot analysis showed that expression of CTGF/Hcs24 mRNA was 3.9 times higher on day 2 of fracture healing than that on day 0. On day 8, it reached a peak of 8.6 times higher than that on day 0. It then declined to a lower level. Immunostaining showed that CTGF/Hcs24 was localized in hypertrophic chondrocytes and proliferating chondrocytes in the regions of regenerating cartilage, and in active osteoblasts in the regions of intramembranous ossification. Although CTGF/Hcs24 was abundant in the proliferating and differentiating cells (on days 8 and 14), immunostaining decreased as the cells differentiated to form bone (on day 20). CTGF/Hcs24 was also detected in cells in fibrous tissue, vascular endothelial cells in the callus, and periosteal cells around the fracture sites. These results suggest that CTGF/Hcs24 plays some role in fracture healing.

Effect of connective tissue growth factor (CCN2/CTGF) on proliferation and differentiation of mouse periodontal ligament-derived cells

BackgroundCCN2/CTGF is known to be involved in tooth germ development and periodontal tissue remodeling, as well as in mesenchymal tissue development and regeneration. In this present study, we investigated the roles of CCN2/CTGF in the proliferation and differentiation of periodontal ligament cells (murine periodontal ligament-derived cell line: MPL) in vitro.ResultsIn cell cultures of MPL, the mRNA expression of the CCN2/CTGF gene was stronger in sparse cultures than in confluent ones and was significantly enhanced by TGF-β. The addition of recombinant CCN2/CTGF (rCCN2) to MPL cultures stimulated DNA synthesis and cell growth in a dose-dependent manner. Moreover, rCCN2 addition also enhanced the mRNA expression of alkaline phosphatase (ALPase), type I collagen, and periostin, the latter of which is considered to be a specific marker of the periosteum and periodontium; whereas it showed little effect on the mRNA expression of typical osteoblastic markers, e.g., osteopontin and osteocalcin. Finally, rCCN2/CTGF also stimulated ALPase activity and collagen synthesis.ConclusionThese results taken together suggest important roles of CCN2/CTGF in the development and regeneration of periodontal tissue including the periodontal ligament.

Gene expression of connective tissue growth factor (CTGF/CCN2) in calcifying tissues of normal and cbfa1-null mutant mice in late stage of embryonic development

Connective tissue growth factor (CTGF/CCN2), one of the most recently described growth factors, is produced by chondrocytes, vascular endothelial cells, and transforming growth factor (TGF)-β-stimulated fibroblasts. CTGF was isolated from a chondrosarcoma-derived chondrocytic cell line, HCS-2/8, and found to be normally expressed in cartilage tissues, especially in hypertrophic chondrocytes, and also to stimulate both the proliferation and the differentiation of chondrocytes in vitro. Therefore, CTGF is thought to be one of the most important regulators of endochondral ossification in vivo. Herein we describe the expression pattern of the ctgf gene in the calcifying tissues of normal developing mouse embryos in comparison with that in core binding factor a1 (Cbfa1)-targeted mutant (cbfa1-null) mouse embryos, in which impaired development and growth were characteristically observed in the skeletal system. After 15 days of development (E15), the expression of ctgf was detected in the zone of hypertrophy and provisional calcification, in which ossification proceeds toward the epiphysis during the skeletal development of the mouse embryo. Furthermore, ctgf was expressed in developing molar and incisal tooth germs around the perinatal stage. However, no expression of the gene was found in the cbfa1-null mouse embryos. These results indicate that CTGF may have certain important roles in the development of the calcifying tissues in the mouse embryo.

Spatial and temporal expression pattern of retinoic acid receptor genes during mouse bone development

Retinoic acid receptor gene; Hybridization, in situ; Bone formation; (Mouse, Forelimb)

Expression of retinoic acid receptor genes in keratinizing front of skin

We found, by an in situ hybridization method with riboprobes synthesized from human cDNA of the retinoic acid receptor (RAR), that the RAR genes (predominantly γ‐subtype) are intensively expressed in the epidermis of normal and psoriasic human skins, and also in keratinizing fronts of 4‐day‐old mouse skins, nail matrices and hair follicles. Thus, target cells of retinoic acid in the skins are concluded to be keratinocytes, which is quite consistent with the fact that retinoic acid regulates keratinization of epidermis in vivo and also modulates expression of the keratin gene in vitro.

Involvement of caspase cascade in capsaicin-induced apoptosis of dorsal root ganglion neurons.

Capsaicin induces apoptosis in some types of neurons, but the exact molecular mechanism remains unclear. In this study, capsaicin was systemically administrated in newborn rats and the dorsal root ganglion (DRG) neurons were examined for caspase-immunoreactivity. Capsaicin-induced neuronal apoptosis was revealed by TUNEL. TUNEL-positive neurons rapidly increased, reaching the peak at 24 h post-injection when 10.6% of DRG neurons were apoptotic. Neurons expressing immunoreactivity for activated caspases-9 and -3 concomitantly increased. At 24 h, 15.9% and 17.7% of DRG neurons exhibited immunoreactivity for caspase-9 and caspase-3, respectively. DNA fragmentation signal and caspase-immunoreactivity were detected in less than 0.5% of DRG neurons of vehicle control rats. The immunoreactivity and TUNEL-positivity returned to the vehicle control level by 120 h. Double label immunohistochemistry revealed co-expression of caspase-9 and DNA fragmentation or caspase-3 and DNA fragmentation. These results suggest that the caspase cascade is involved in the primary neuronal apoptosis induced by neurotoxin capsaicin.

Expression of Connective Tissue Growth Factor/Hypertrophic Chondrocyte-Specific Gene Product 24 (CTGF/Hcs24/CCN2) During Distraction Osteogenesis

To investigate the localization and expression of connective tissue growth factor/hypertrophic chondrocyte-specific gene product 24/CCN family member 2 (CTGF/Hcs24/CCN2) during distraction osteogenesis in the rat femur, we studied a total of 54 male rats (11 weeks old). We performed osteotomy in the midshaft of the right femur. After 7 days (lag phase), distraction was started, at the rate of 0.25 mm/12 h for 21 days (distraction phase) by using a small external fixator, and this was followed by a 7-day consolidation phase. Localization and expression of CTGF/Hcs24 during distraction osteogenesis in the femur were examined by immunostaining, in situ hybridization, and reverse transcriptase polymerase chain reaction (RT-PCR). Immunostaining showed the localization of CTGF/Hcs24 in various cells located in the bone-forming area around the osteotomy site. During the distraction phase, in situ hybridization showed that CTGF/Hcs24 mRNA was expressed not only in hypertrophic chondrocytes and osteoblasts but also in fibroblast-like cells and mesenchymal cells at sites of end-ochondral ossification, and not only in osteoblasts but also in pre-osteoblasts and fibroblast-like cells at sites of intramembranous ossification. RT-PCR showed higher level expression of CTGF/Hcs24 mRNA in the distracted group than in the nondistracted group. These results revealed an elevated pattern of CTGF/Hcs24 mRNA expression during distraction osteogenesis, and suggest that CTGF/Hcs24 may play some roles in the endochondral and intramembranous ossification processes that occur during distraction osteogenesis.

Tyrosine kinase type-receptor ErbB4 in chondrocytes : interaction with connective tissue growth factor and distribution in cartilage

In order to identify receptor molecules that participate in the growth and differentiation of chondrocytes, we cloned a number of cDNA fragments from HCS‐2/8 chondrocytic cells, by using tyrosine kinase‐specific primers for amplification. The mRNA expression of one such receptor, ErbB4, was increased by connective tissue growth factor/hypertrophic chondrocyte‐specific gene product (CTGF/Hcs24), which promotes all stages of the endochondral ossification in vitro. ErbB4 expression was observed through all stages of chondrocytic differentiation in vitro, corresponding to the wide distribution of CTGF/Hcs24 target cells. Furthermore, positive signals for erbB4 mRNA were detectable throughout most populations of chondrocytes, in growth and articular cartilage in vivo. These results demonstrate for the first time that ErbB4 is expressed in chondrocytes and may play some roles in chondrocytic growth and differentiation along with CTGF/Hcs24.