:;,.1/+/%
*$<
= "59#
2<
=
2007 ! 2 ' 17 &>25 &
7)
34-80 hematological- and neurological expressed sequence 1 6(
Department of Earth and Space Science,
Graduate School of Science, Osaka University
Assistant Professor: Tatsushi Goto
Ohio State University, Dayton University and Miami University (Ohio, USA)
Feb. 17 - Feb. 25, 2007
Oral presentation
“Analyses of hematological- and neurological expressed sequence 1 during newt retina regeneration.”
In the presentation in Dayton University
International Internship Report
21st COE Program “Toward A New Basic Science : Depth and Synthesis”
Comparing the variety of animals is a basic idea to understand the evolution of life on the earth. I
have examined the newt retina regeneration for several years. To develop our research, we think that it
is necessary for us to learn how the research on other vertebrates are done. This time, I visited 3
researchers; Dr. Fischer (Ohio State University), Prof. Tsonis (Dayton University) and Dr. Tsonis
(Miami University), which are studying the eye regeneration in various vertebrates. I had the oral
presentation “Analyses of hematological- and neurological expressed sequence 1 during newt retina
regeneration.” in Dayton University and Miami University as follows:
Our primary reason to study the newt retinal regeneration is because it is amazing that they can
always regenerate their retina, despite that retinas can be done during a restricted stage in early
development in most vertebrate. We are interested in their high ability of tissue regeneration that was
lost in the evolution of human. Secondary, we think that the retinal regeneration of newt is a good
model to analyze the various stage of the neural tissue formation. Also, we think this system has an
advantage of being able to restrict the effect from other tissues. Based on these points, we are
investigating the molecular mechanism of newt retinal regeneration.
After the retinal removal, the remaining RPE cells lose their pigment granules, which is called
dedifferentiation of RPE cell. They form an ‘early’ regenerating retina with 1-2 cells thickness, which
are comprised pigmented cells and non-pigmented progenitor cells. In the next ‘intermediate’ stage,
the retinal progenitor cells proliferate. Next, the redifferentiation into the retinal neurons begins. In the
‘late’ regenerating retina, the synaptic layers are formed and the retinal morphology is restored like
original one. Some experiments using retinal cell-specific markers suggest that the cell differentiation
during the retina regeneration recapitulates that during the retina development. Although newts have
retinal stem/progenitor cells in the ciliary marginal zone to append neurons to the existing retinas in
adults, the dedifferentiated RPE cells are main contributors to form retinas in this case.
Dedifferentiation of RPE cells is an unique and important event for newt retina regeneration. However,
the molecular mechanism of the process remains to be elucidated.
We examined the gene expression pattern at the early regeneration stage by differential display
method and found that some genes upregulated or downregulated within a few days after the
retinectomy in newts. One of such genes encodes newt Hn1 gene. The previous reports indicate that
Hn1 gene is involved in the development and the regeneration of neuronal tissues. But the Hn1 genes
except for mammals has not been investigated. In addition, a known functional motif is not contained
in the sequence. Therefore, it is not understood about its function. I think it is necessary to study Hn1
gene more.
Our molecular phylogenetic analysis showed that vertebrates generally have a Hn1 gene and its
homologous genes. We examined the tissue distribution of Hn1 mRNA by RT-PCR, so that Hn1
mRNA existed more abundantly in the retinas than in other tissues. After the retinectomy in mice, the
transcription level of mouse Hn1 didn’t change so much. It has been proposed that the upregulation of
Hn1 occurs in the neuronal system that is able to regenerate. This results support this hypothesis.
Next, we prepared recombinant HN1 protein, and the antiserum raised against it to examine where
the HN1 proteins localize in newt retina. The HN1 was localized in the outer plexiform layer, the inner
plexiform layer and the ganglion cell layer in normal (unoperated) retinas. After retinal removal, the
HN1 was induced in the differentiating RPE cells. Then, HN1 localized in the retinal progenitor cells.
Proceeding the retinal regeneration, the localization of HN1 changed like and finally restored the
pattern of normal retina. Retinal progenitor cells are also present in eyes of developmental embryos,
and we performed immunohistochemical analyses of HN1 in the developmental retina to compare
with the regenerating retinas. It is indicated that the localization pattern of HN1 in the developmental
retina was similar to that in the regenerating retina. In addition, our immunohistochemical analyses
indicated that the subcellular localization patterns of HN1 protein. In well-differentiated tissues, the
HN1 protein resides in the cytosol of the neurons. In undifferentiated tissues, however, the HN1 was
induced and localized not only in the cytosol but also in the cell nuclei. Although this is only a guess,
it is possibly that HN1 travels between the cytosol and the cell nuclei with a long cycle.
In conclusion, newt Hn1 is upregulated and possibly plays an undefined role in early retina
generation. However, there have been only a few studies of Hn1 and many questions remain to be
answered. We should do mutagenetic, cellular biological, and biochemical analyses both in vitro and
in vivo. Now we are performing or planning to examine these points about Hn1 gene and hope to reply
to the question what the function of Hn1 is as early as possible.
I thank my collaborators; Prof. Tokunaga, Dr. Hisatomi and Ms. Kotoura in Osaka University; and
Prof. Saito and Dr. Chiba in Tsukuba University. I have gratefully appreciated their help on this work.
Finally, I also appreciate the 21st COE Program “Towards a New Basic Science: Depth and
Synthesis” for my support.
Refereces
[1] Goto et al., Experimental Eye Research 83 (2006): 972-980.
[From left]
Dr. Nakamura (Dayton University), Dr. Tsonis
(Miami University) and me.