DIVISION OF CELL DIFFERENTIATION


                              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.

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.

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.

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.