NATIONAL INSTITUTE FOR BASIC BIOLOGY
DIVISION OF CELL MORPHOGENESIS
- Professor:
- 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.
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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.
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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.
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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.
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- 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.
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- 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.
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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.
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- 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.
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- 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.
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- 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.
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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.
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Publication List:
- Eguchi, G. and Kodama, R. (1993) Transdifferentiation. Curr. Opin. Cell
Biol. 5, 319-325.
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- 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.
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- 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.
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- 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.
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- 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.
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- 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.