NATIONAL INSTITUTE FOR BASIC BIOLOGY
DIVISION OF CELL DIFFERENTIATION
- Professor:
- Yoshiaki Suzuki
- Associate Professor:
- Kohji Ueno
- Research Associates:
- Shigeharu Takiya
Toshiharu Suzuki
Kaoru Ohno
- JSPS Postdoctoral Fellows:
- Kazuhito Amanai
Vaclav Mach
- Visiting Scientists:
- Masakazu Fukuta (from Aichi University of Education)
Pin -Xian Xu
- Graduate Students:
- Kaoru Ohno (Graduate University for Advanced Studies)
Pin-Xian Xu (Graduate University for Advanced Studies)
Hiroki Kokubo (Graduate University for Advanced Studies)
Xin Xu (Graduate University for Advanced Studies)
- Technical Staffs:
- Miyuki Ohkubo
Chikako Inoue
Members of the Division have been involved in two well associated projects. One
, which was initiated in 1968, is to understand how a special tissue like the
silk gland of Bombyx mori is differentiated along the developmental programs
and results in transcribing a specific set of genes like the silk fibroin,
fibroin L-chain, P25, sericin-1, and sericin-2 genes. The other initiated at
the time when the Division was established in 1978 is concerned with how the
body plan of the silkworm is controlled and how the developmental regulatory
genes regulate a set of target genes in specifying the identities of various
regions of the embryos.
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I. Genes and factors that control the silk gland development and
the silk genes transcription
- We have been trying to understand the networks of gene regulation
hierarchy that function in the processes of silk gland development and
differentiation. As a bottom-up type approach for this project, analyses
on the molecular mechanisms that control the differential transcription
of the fibroin and sericin-1 genes in the silk gland should shed light
on a part of the networks. In complementing this approach, a top-down
type approach should also help understanding the networks; analyses of
regulation hierarchy of the homeobox genes and other regulatory genes,
and identification of their target genes expressed in the labial
segment, where the silk gland is originated.
-
- Among many factors proposed to bind and control the fibroin and
sericin-1 genes, the POU-MI which accommodates a POU-domain identical
to Drosophila Cf1-a was cloned previously and characterized. The POU-MI
binds to the SC region of the sericin-1 gene and is assumed to enhance
the transcription. This protein also binds to the PB element of the
POU-MI gene and suppreses the transcription. The expression of the
POU-MI gene has been analyzed in Bombyx embryos by in situ
hybridization. To our surprise, the gene was expressed specifically for
the first time at stage 18 19
(Fig. 1C) in the limited site of the
labial segment where the silk gland is going to be formed by
invagination
(Fig. 1A). This
location exactly matches with the site
where the Bombyx Scr expression disappears specifically
(Fig. 1B) which
was detected in the entire labial segment in the preceding stage (see
the section II). The POU-MI expression continues along with the silk
gland development and is confined to the middle portion of the silk
gland by late embryonic stages. It was late in the stage 22 when the
POU-MI expression was also detected in the central nervous system as
expected for a Cf1 -a homologue. These observations suggest that the
POU-MI gene may have multiple functions; (1) contribution to the
commitment of the primordial silk gland cells, (2) roles in maintenance
of the silk gland development, (3) roles in establishing terminally
differentiated states as a positive transcription factor for the
sericin-1 gene and a negative transcription factor for its own gene,
and (4) contribution to the commitment of the nerve cells.
-
- Silk gland specific transcription factor SGF-1 interacts with the SA,
FA and FB sites localized upstream of the sericin-1 and fibroin gene
promoters. Partial purification of this factor was achieved using SA
site-DNA affinity resin. Competitive gel shift assay using proteins
renatured from SDS-PAGE showed that SGF-1 corresponds to two
polypeptides migrating on 42 and 43 kDa, respectively. In the next
purification step, these two proteins were recovered in a single RPC
fraction of more than 80 percent Purity. The 42 and 43 kDa proteins
were digested directly in SDS-PAGE gel, and peptides were eluted,
fractionated and sequenced. Four of them yielded useful sequences,
which should facilitate molecular cloning of SGF-1 gene. The pure 42
and 43 kDa proteins were used to study the SA binding site by the
methylation interference assay
(Fig. 2). Their footprints appear
identical with each other and with the footprint obtained from the
specific retarded band produced by the SA probe in a crude extract.
-
- Intronic modulator (enhancer II; +156/+454) of the fibroin gene is
composed of 6 octamer-like elements. The octamer binding factor-1
(OBF-1) binds to the elements at around +220 and +290 in the enhancer
II, and also to the element at around 130 in the upstream enhancer
(enhancer I). The OBF-1 activity was detected only in the posterior
silk gland abundantly at stages only when the fibroin gene was
expressed. Purification of OBF-1 is in progress and 32 kDa protein
recovered from SDS-PAGE showed the OBF-1 activity. The second octamer
binding factor (OBF-2) binds strongly to the element at around +420 and
weakly to other elements at around +220, +290 and +370. The OBF-2 binds
also to the enhancer I weakly. Gel shift assay using a POU-MI specific
antibody showed that the OBF-2 is the POU-M1. The third octamer binding
factor (OBF-3) binds to the elements at around +220 and +290, and also
to an element at around -60. An oligonucleotide corresponding to the
+290 element competed the transcription enhancement both by the
enhancer I and II in the posterior silk gland extracts. Integration of
these factors as well as FF1 and 2 which were purified before will be
important for regulation of the fibroin gene transcription.
-
II. Genes involved in the Bombyx body plan
- We have isolated a caudal (cad) homologue from a cDNA library of Bombyx
mori embryos. The Bombyx cad transcripts were firstly accumulated in the
nurse cells and transferred into the oocyie in a definite period during
oogenesis. The maternal transcripts formed a concentration gradient
spanning anteroposterior axis during the gastrulation stage and were
restricted to the anal pad after 2 days of embryogenesis (Development
120, 277 285 (1994)). This observation gives a sharp contrast with the
Drosophila cad expression pattern which reveals the corresponding
expression profile during the syncytial blastoderm stage. The Bombyx
cad protein was not detected in the ovary and early 9 hrs of eggs, but
was first detected evenly during cellular blastoderm stage. It was
during gastrulation when Bombyx cad protein concentration gradient
shifted along the anteroposterior axis. The observed distinct timing
and conservation on mRNA as well as protein gradients formation between
Drosophila and Bombyx might contribute to realize differences in the
body plans and give some clues to elucidate the mechanism and function
related to mRNA and protein concentration gradients.
-
- To understand how the labial segment identity is determined and the
silk gland development is controlled, we have begun characterizing
expression patterns of Bombyx Sex combs reduced (Scr), Deformed (Dfd),
fork head (fkh), and POU-M1. The Bombyx Dfd was expressed in the
mandibular and maxillar segments but not in the labial segment where
the Bombyx Scr was specifically expressed. As described in the previous
section, the Bombyx Scr expression was eliminated in the invagination
site where the silk gland development takes place. This elimination was
complemented with the specific expression of POU-M1. During the silk
gland development the POU-MI expression was detected in the entire
region of the gland in the early phase, and restricted to the anterior
portion and middle portion of the gland and finally to the middle
portion, while the Bombyx fkh expression was detected in the middle and
posterior portions in the middle phase of development. These
observations may outline the framework of the hierarchy in the silk
gland development and differentiation.
-
- In continuation of the abdominal segments identification, we have
concentrated in the study of morphogenesis of embryonic abdominal legs.
We have analyzed proteins in the wild type embryos by SDS-PAGE, and
found that a 270 kDa protein (p270) is expressed specifically in the
abdominal legs. We have purified the p270 from embryos, and prepared a
specific antibody against the p270. Using the antibody no p270 was
detected by Western blot analysis in the homozygous E(Ca)/E(Ca) embryos
which do not carry the Bombyx abd-A gene. It is likely that the p270
expression is under the control of the Bombyx abd-A gene.
Immunohistochemical analysis indicated that the p270 is localized in
restricted cells of the wild type abdominal legs but not detected in
the late embryonic stages. To study a role of the p270 in morphogenesis
of embryonic abdominal legs, molecular cloning of p270 cDNA is being
planned.
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Publication List:
- Fukuta, M., Matsuno, K., Hui, C.-c., Nagata, T., Takiya, S., Xu, P.-X.,
Ueno, K. and Suzuki, Y. (1993) Molecular cloning of a POU
domain-containing factor involved in the regulation of the Bombyx
sericin-1 gene. J. Biol Chem. 268, 19471-19475.
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- Takiya, S. and Suzuki, Y. (1993) Role of the core promoter for the
preferential transcription of fibroin gene in the posterior silk gland
extract. Develop. Growth Differ. 35, 311 321.