Goro Eguchi

Associate Professor:
Ryuji Kodama

Research Associate:
Makoto Mochii

JEPS Postdoctoral Fellow:
Keiko Ishikawa

Visiting Scientists:
Takamasa S. Yamamoto
Kaichiro Sawada
Christine Baader (from Zoological Institute, University of Basel)

Graduate Students:
Akio Iio (Graduate University for Advanced Studies)
Nobuhiko Mizuno (Graduate University for Advanced Studies)
Yuuichi Mazaki (Graduate University for Advanced Studies)
Takeshi Kitagawa (from School of Medicine, Nagoya University)
Jatupol Kosittsawat (from School of Medicine, University of Tokyo)
Yasutaka Matsubara (from Shinshu University)

Visiting Researcher:
Hiroyuki Horiuchi (Research Associate, Faculty of Agriculture, Hiroshima University)

Technical Staffs:
Chiyo Takagi
Hisae Urai

There is a mode of reparative regeneration, in which the lost tissue or organ can be compensated by cellular metaplasia (transdifferentiation) of once specialized tissue cells. In the newt and some other limited species of the vertebrate, the lens and neural retina can be completely regenerated through the transdifferentiation of pigmented epithelial cells (PECs). Such a phenomenon, transdifferentiation, as observed in regeneration of ocular tissues seems to be a highly powerful model for studying stability and instability in differentiation of tissue cells. From this view point, lens transdifferentiation of PECs of vertebrates has been studied in in vivo and in vitro systems, and our in vitro studies have revealed that dormant potential to transdifferentiate into lens cells is widely conserved throughout vertebrate species including human.

Our studies have been conducted to clarify the molecular mechanism controlling the lens transdifferentiation of PECs and also to search the reason why the pigmented epithelia of species other than the newt and so forth never regenerate the lens in the in situ eyes. Based on findings accumulated up to the last year, we have conducted analysis of the lens transdifferentiation, particularly focussing on genes which have been predicted to have essential roles in regulation of the differentiated state of PECs and also of lens transdifferentiation of them. The followings are abstracts of investigations and results obtained in 1993 through studies of these major projects and the following additional subjects which have been conducted as collaborative works with scientists from the outside: (1) Pattern formation of Lepidopteran wing and (2) Basic analysis of biocompatibility of hydroxyapatite as a biomaterial.

I. Analysis of functions of genes responsible for the lens transdifferentiation of PECs

It has been predicted by analysis thus far that products of two genes, tentatively designated as pP344 and pP64 must function to regulate the differentiated state and lens transdifferentiation of PECs in vivo and in vitro. In this year, we have extended our study to analyze functions of these two genes. We analyzed pP344 gene expression during chicken eye development by RT-PCR and in situ hybridization and also characterized the pP344 protein using antipeptide antibodies. The time course of expression level showed two peaks; the first peak occurred around the 10th day similarly to the expression of melanosome-related genes, while the second peak occurred just after hatching when PECs had completely differentiated, suggesting that pP344 gene may be related to the function of fully differentiated PECs. Anti-synthetic peptide antibodies detected pP344 protein in the culture medium of the PECs but not within the cells, strongly suggesting that pP344 gene product is a secreted protein. The pP64 gene produces two different transcripts, 5.0kb and 6.0kb mRNAs, whose products are TGFB-binding proteins, and fully differentiated PECs express 5.0kb mRNA as a major product in addition to 6.0kb mRNA but both multipotent dedifferentiated PECs and transdifferentiated lens cells express only 6.0kb mRNA. It has been clearly shown that protein produced by 5.0kb mRNA is secreted by PECs but protein produced by 6.0kb mRNA is trapped by the extracellular matrix of PECs in the in situ eye. These results must be the fundamental information for our further studies on the molecular regulation of the lens transdifferentiation of PECs.

II. Analysis of transcriptional regulator in pigmented epithelial cells

The product of mouse microphtalmia (mi) gene is thought to be a class of basic helix-loop-helix-zip transcriptional factor which may regulate the directions of differentiation of some types of cells including pigmented epithelial cells, because some mutations in mi gene cause transformation of pigmented epithelium to neural retina in mice. To analyze the molecular mechanisms in differentiation and transdifferentiation of cultured pigmented epithelial cell, avian homologs of mi gene were isolated from cDNA libraries of chicken and quail pigmented epithelial cells. Nucleotide and amino acid sequences of mi are well conserved between aves and mammals. The products of mi genes have a basic helix-loop-helix-zip domain similar to ubiquitous transcriptional regulators TFE3, TFEB and TFEC, showing possible interactions of mi product and some factors relating the TEFs in gene regulation. Northern blotting shows activation of mi gene during differentiation of pigmented epithelial cells and inactivation of it in dedifferentiated state of cells suggesting the key role of mi gene in differentiation of pigmented epithelial cells. The possibility in which transdifferentiation of pigmented epithelial cells may be regulated by activity of mi product is now analyzed by genetic manipulation of mi gene in cultured cells.

III. Expression of connexin in the pigmented epithelial cells

The morphological change of the gap junction in the course of the transdifferentiation of PECs of the chick embryo was previously studied. In essence, although the gap junction is abundantly found in the PECs cultured in vitro, it is temporally lost in the dedifferentiated PECs but reappear in the lentoids or redifferentiated PECs. Molecular biological analysis showed that the gap junction in the PECs is made up of chicken connexin 43, which is one of the genes for the main component of the gap junction. We are preparing probes for other connexin genes in order to clarify the expression pattern of connexin genes in the course of transdifferentiation. Here we summarize some preliminary data on connexins.

Polyclonal antibodies against connexin 43 were raised against synthetic peptides. One of the antibody against a cytoplasmic loop reacted specifically and can serve as a good marker. Another antibody against an extracellular loop reacted from outside of the cell. This reaction was shown by directly applying the antibody to living cells, and then detecting the bound antibody with fluorescentlabeled second antibody. This result suggests that it may be possible to modify the gap junctional cell-to-cell communication from outside of the cell, thus enabling the inhibition of gap junction in large population of cultured cells.

The pattern of connexin 43 gene expression was examined in the eyes of the 9-day-old chick embryo by in situ hybridization technique. Although the expression is seen throughout the pigmented epithelium, the expression is conspicuously strong in the iris and the ciliary pigmented epithelium. The immunofluorescent staining with anti-connexin 43 peptide antibody also showed high level of connexin 43 protein in the iris and ciliary pigmented epithelium. The expression pattern of a PEC-specific gene, pP344, which was found in our laboratory, showed an opposite gradient of expression, i. e. highest expression is seen in the retinal PE. These genes can be excellent markers for the regional specificity of the pigmented epithelium and also it is possible that they bear some roles in the physiological activity of each epithelium.

IV. Analyses of the process of programmed cell death in the pupal wing of Pieris rapae

The shape of the wings of butterflies are formed by an extensive cell death at the margin of the wings during the pupal period. We have suggested, through ultrastructural observations of the pupal wings of Piris rapae, that the cell death observed here resemble the apoptosis, which is a characteristic form of programmed cell death reported to occur throughout the development of vertebrates and invertebrate.

One of the critical symptom of the apoptosis is the fragmentation of the DNA in the nucleus prior to the death, but examination of such fragmentation by standard method using the electrophoresis must be difficult because the amount of the tissue is very limited. We recently adopted the TUNEL method (TdT-mediated dUTP-biotin nick end labelling) to detect the fragmentation of DNA in in situ tissue. This procedure adds biotinylated dUTP at existing 3' ends by an enzyme terminal deoxynucleotidyl transferase. The nuclei containing DNA with many breaks has much more number of 3' ends than other nuclei, so that much more biotinyl residues are incorporated. The biotinyl residue is detected by avidin-biotinyl-horse raddish peroxidase complex (ABC) yielding staining on the nuclei with DNA fragmentation. We applied this method to the whole mount preparation of the pupal wing and showed that nuclei with positive staining are observed at the period and the area of extensive cell death formerly observed ultrastructurally (Fig.). Although this method provides no information on the length of the DNA fragments, this result strongly suggests that there works a mechanism of cell death including DNA fragmentation resembling the apoptosis in the process of the formation of the wing of butterflies.

We also re-examined ultrastructural data and concluded that cells with a morphology of macrophages are abundantly seen near the area of cell death, suggesting that the dead cells are actively phagocytosed by these macrophages.

We have concluded at present that the loss of the margin of the pupal wing proceeds by the programmed cell death of the epithelial cells at the margin and by the removal of cell debris through phagocytosis by neighboring epithelial cells and by macrophages. The molecular mechanism determining the area to be removed and how this determination is realized as cell death are two important aspects of further studies.

V. An attempt to improve biocompatibility of hydroxyapatite as a biomaterial

Based on findings of cell biological analysis conducted thus far, we have attempted to improve biocompatibility of hydroxyapatite using in vitro culture system of human gingival cells established in our laboratory. Adhesion, spreading and growth of gingival cells, epithelial cells, and connective tissue fibroblasts, cultured on the hydroxyapatite can be dramatically enhanced when the hydroxyapatite surface is modified by coating with type I collagen molecules after ionetching under condition of 10^(-2) mmHg with an ion-sputtering equipment, suggesting a strong possibility to improve biocompatibility of hydroxyapatite materials by biochemical modification of its surf ace.

Publication List:

Eguchi, G. and Kodama, R. (1993) Transdifferentiation. Curr. Opin. Cell Biol. 5, 319-325.

Hyuga, M., Kodama, R. and Eguchi, G. (1993) Basic fibroblast growth factor as one of the essential factors regulating lens transdifferentiation of pigmented epithelial cells. Int. J Dev. Biol. 37, 319-326.

Agata, K., Kobayashi, H., Itoh, Y., Mochii, M., Sawada, K. and Eguchi, G. ( I 993) Genetic characterization of the multipotent dedifferentiated state of pigmented epithelial cells in vitro. Development 118, 1025-1030.

Sawada, K., Agata, K., Yoshiki, A. and Eguchi, G. (1993) A set of anti-crystallin monoclonal antibodies for detecting lens specificities: B-crystallin as a specific marker for detecting lentoidogenesis in cultures of chicken lens epithelial cells. Jap. J. Ophthalmol. 37, 355-368.

Orii, H., Agata, K., Sawada, K., Eguchi, G. and Maisel, H. (1993) Evidence that the chick lens cytoskeletal protein CP49 belongs to the family of intermediate filament proteins. Curr. Eye Res. 6, 583-588.

Araki, M., Kodama, R., Eguchi, G., Yasujima, M., Orii, H. and Watanabe, K. (1993) Retinal differentiation from multipotential pineal cells of the embryonic quail. Neurosci. Res. 18, 63-72.