Eiki Koyama

Expression of hepatocyte growth factor gene in endothelial and Kupffer cells of damaged rat livers, as revealed by in situ hybridization

Hepatocyte growth factor (HGF) has been demonstrated to be synthesized and secreted by non-parenchymal liver cells for liver regeneration after hepatic injury. We performed in situ hybridization to identify HGF-producing cell types in rat liver hepatitis induced by administrating carbon tetrachloride as a hepatotoxin. We found that transcripts of the HGF gene are localized in the Kupffer and endothelial cells in normal livers and increased remarkably in the Kupffer cells of the damaged livers. Thus, HGF is concluded to be synthesized in the Kupffer and endothelial cells to repair the liver tissue in paracrine fashion. No significant increase in the transcripts of the HGF gene was observed in livers after partial hepatectomy, indicating that a mechanism on liver regeneration after the hepatectomy differs from that on liver repairs. Since the HGF gene expression was also found in lung and kidney, HGF may be a ubiquitous factor for tissue repairs.

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 retinoic acid receptor genes in neural crest-derived cells during mouse facial development

Retinoic acid (RA) is known as a teratogen that induces abnormalities in facial structures which are made up mainly of neural crest‐derived mesenchyme. We investigated expression patterns of RA receptor (RAR) genes (subtypes α, β, γ) during mouse facial development. The expression of the rarβ gene is specific for the mesenchyme around developing eyes and nose, whereas the RARγ gene is expressed in the mesenchyme differentiating to facial cartilages and bones. In contrast, the RARα gene is expressed weakly and uniformly over the facial region. These results suggest that crucial roles of endogenous RA in facial development depend on differential functions of the RAR subtypes.

Bacterial endotoxin-induced expression of metallothionein genes in rat brain, as revealed by in situ hybridization

In order to clarify acute-phase response in brain, we investigated induction of metallothionein (MT) genes by administrating an endotoxin (lipopolysaccharide) in rat intraperitoneum. We performed in situ hybridization on the serial brain sections to identify the cells expressing the MT genes in acute-phase. After endotoxin administration, transcripts of MT genes were detected in the arachnoideal, ependymal cells and glial cells around the Purkinje cells of the cerebellum, while no significant induction of the MT genes by zinc ion was observed in brain. These results suggest that the acute-phase response occurs specifically in at least these 3 non-neuronal cells.

Expression pattern of the activin receptor type IIA gene during differentiation of chick neural tissues, muscle and skin

To elucidate target cells of activins during embryogenesis we isolated cDNAs of chick activin receptor type II (cActR‐II) and studied expression patterns of thecActR‐II gene by in situ hybridization. Transcripts ofcActR‐II were observed in neuroectoderm developing to spinal cord, brain and eyes, in surface ectoderm differentiating to epidermis, and in myotomes differentiating to muscles. The expression patterns ofcActR‐II suggest that activin and its receptor are involved in differentiation of chick neural tissues, muscle and skin after inducing the dorsal mesoderm.

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.

Retinoic Acid Treatment Induces the Ectopic Exporession of Retinoic Acid Receptor β Gene and Excessive Cell Death in the Embryonic Mouse Face

Maternal treatment with 100 mg/kg of retinoic acid (RA) on day 9 of gestation in mice caused craniofacial abnormalities of the mandibulofacial dysostosis type. The abnormal morphology was attributed to the excessive cell death in the dorsal aspects of the maxillary and mandibular prominences of the first pharyngeal arch and in the proximal region of the mandibular prominence. To investigate the expression of the RA receptor (RAR) genes in abnormal face morphogenesis, in situ hybridization was performed. The distribution patterns of RAR α and γ transcripts were not altered in the treated embryos. By contrast, the teratogenic dose of RA increased the level of RAR β transcripts, as early as 3 hr after RA‐treatment, in the regions where the RAR β expression is at a low level in normal development. The increase of RAR β transcripts was detected by 12 hr, and declined to the low level within 24 hr after the treatment. The regions where ectopic expression of RAR β gene was observed included the areas where the excessive cell death occurred 9–12 hr after RA‐treatment. These results suggest that ectopic induction of RAR β by RA may lead to the excessive cell death, threfore may cause abnormal morphogenesis.

Involvement of retinoic acid and its receptor β in differentiation of motoneurons in chick spinal cord

Retinoic acid is known to play an important role during development of central nervous system. In order to clarify function of retinoic acid during the development, we investigated expression pattern of the chick retinoic acid receptor subtype beta gene by an in situ hybridization method. We found that expression of the beta gene is localized in neural tube at stages 16-20, then is turned to be restricted to developing motoneurons at stages 23-29. These results suggested that retinoic acid and its receptor beta are involved in differentiation of the motoneurons in spinal cord.

Cooperative Activation of HoxD Homeobox Genes by Factors from the Polarizing Region and the Apical Ridge in Chick Limb Morphogenesis

When a mouse zone of polarizing activity (ZPA) at the posterior margin of the limb bud was grafted into the anterior margin of the chick limb bud, the expressions of the chick homeobox genes HoxD12 and D13 were induced prior to the formation of chick extra digits. This induction was observed in a restricted domain close to both the grafted mouse ZPA and the chick apical ectodermal ridge (AER). When the posterior half of the AER was removed, the normal expression was diminished in the distaloposterior region. Thus, it is likely that at least two distinct factors, one from the ZPA and the other from the AER, act cooperatively to provide positional information to induce the sequential expression of the HoxD genes.