National Insitute for Basic Biology

DIVISION OF CELL DIFFERENTIATION

Professor:
Yoshiaki Suzuki
Associate Professor:
Kohji Ueno
Research Associates:
Shigeharu Takiya
Toshiharu Suzuki
Kaoru Ohno
Hiroki Kokubo
JSPS Postdoctoral Fellow:
Vdclav Mach
Visiting Scientists:
Pin-Xian Xu
Xin Xu
Graduate Students:
Hiroki Kokubo (Graduate University for Advanced Studies)
Xin Xu (Graduate University for Advanced Studies)
Yoshinori Ueno (Graduate University for Advanced Studies)
Katsuyoshi Matsunami (Graduate University for Advanced Studies)
Technical Staffs:
Miyuki Ohkubo
Chikako Inoue
Members of the Division have been involved in two well associated projects. One 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.

l. 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-l 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-M1 which accommodates a POU-domain identical to Drosophila Cf1-a was cloned previously and characterized. The POU-M1 binds to the SC site of the sericin-1 gene and is assumed to enhance the transcription. This protein also binds to the PB element of the POU-M1 gene and suppresses the transcription. The expression of the POU-M1 gene has been analyzed in Bombyx embryos by in situ hybridization and immunohistochemistry. The gene was expressed for the first time at stage 18-19 in a restricted site of the labial segment; the primordial cells of prothoracic gland. This location matches with the site where the Bombyx Scr expression disappears which was detected in the entire labial segment in the preceding stage. Later the POU-M1 expression is observed along with developing silk gland and is confined to the anterior and middle portions of the silk gland by late embryonic stages. These observations suggest that the POU-M1 gene may have multiple functions.

Silk gland specific transcription factor SGF-1 interacts with the SA site of the sericin-1 gene and FA and FB sites of the fibroin gene. Two related SGF-1 polypeptides were purified of observed molecular weights 40 kDa and 41 kDa (V. Mach et al.(1995). J Biol. Chem., in press). These proteins interact specifically with the SA site in electrophoretic mobility shift assay and DMS methylation interference assay. The SGF-1 40 kDa protein was partially sequenced and found to be a new member of the fork head/HNF-3 family. This information facilitated cloning of several full length cDNAs coding for the SGF-1 40 kDa and possibly also for the SGF-1 41 kDa proteins (V. Mach et al. (1995). J Biol. Chem., in press). Sequencirrg of the full length cDNAs revealed three domains conserved between Drosophila Fork head protein and mammalian HNF-3 factors (Fig. 1). SGF-1 mRNA is expressed consitently with the presumed role of its protein product in the regulation of sericin-1 gene and SGF-1 protein contains putative transactivation domains. We conclude that regulation of the sericin-1 gene transcription via SA site is exerted by the SGF-1 40 and 41 kDa proteins.

Fig.1

Fig.1 Comparison of SGF-1 with other members of Fork head/HNF-3 family.
The three domains conserved between Drosophila Fork head and mammalian HNF-3 factors are present also in SGF-1. The domain boundaries in SGF-1 are Lys103/Glu212(I), Leu286/Leu303(II), and Asp331/Leu349(III). Domain I is the DNA binding domain, and domains II and III participate in transactivation by HNF-3 beta.

The expression patterns of SGF-1 mRNA and its protein were analyzed in embryos. The transcripts and protein were detected firstly in the most anterior and posterior domains at the time when the germ anlage is formed, and at later stages in the entire region of the foregut, in the most anterior and posterior regions of the elongated midgut, and in the entire region of the hindgut (Fig. 2A). At the time when the embryo retraction is finished (stage 20) the transcripts and protein are also detected in the invaginated silk gland (Fig. 2A). By the time when the blastokinesis is finished (stage 25) the transcripts and protein are restricted to the middle and posterior regions of the silk gland (Fig. 2B). These results suggest that the Bombyx fork head/SGF-1 might play important roles in the gut and silk gland development.

Cloning and expression pattern analysis of the Bombyx Scr have been described previously. Regulation of the Bombyx Scr gene itself in the labial segment and search for the target genes of the Bombyx Scr have been intiated.

Fig.2

Fig.2 Expression of Bombyx Fork head/SGF protein.
A. An embryo at the embryo retraction stage (stage 20). The triangles from left to right indicate positive signals at the foregut, the anterior most of the midgut, the posterior most of the midgut, and the hindgut, respectively. The arrowheads indicate the signals at the invaginated silk glands.

B. A silk gland dissected out from an embryo of stage 25. The arrowhead indicates the border between the anterior (negative) and the middle (positive) silk gland. The positive signal continues all the way to the end of the posterior silk gland.

II. Other genes of developmental interest

Following the previous discovery that the Bombyx cad transcripts and protein form concentration gradients at different timing of development than in Drosophila, our attention has been forwarded to Bombyx pair-rule genes. We have cloned the Bombyx even-skipped and Bombyx hunchback cDNAs. After examining the expression patterns of these genes, we propose a possible molecular explanation for Bombyx segmentation mechanism. At the top of interests, our data suggest that Bombyx even-skipped might serve a doublesegment defining role and determine on the odd-numbered Bombyx engrailed stripes.

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 two high molecular weight proteins of 270 and 260 kDa (p270/260) are expressed specifically in the abdominal legs. These proteins are not detectable in the ECa/ECa embryo which lacks Bombyx abd-A gene as reported previously, suggesting a control under the Bombyx abd-A. We have purified the p270/260 to greater than 90% homogeniety by ammonium sulfate fractionation, hydroxyapatite column chromatography and gel filtration column chromatography. Since p270 and p260 were coeluted through these procedures, we assume that p270 and p260 participate in the formation of a stable complex.

Immunohistochemical analysis demonstrated that p270/260 was detected in a few cells in each abdominal leg anlage of the first to the eighth abdominal segments at an early developmental stage. The number of positive cells increased and they were integrated into abdominal legs of the third to the sixth abdominal segments in later stages. From these results we speculate that p270/260 may have an important role in the development of abdominal legs.

Publication List:

Amanai, K., Suzuki, Y. and Ohtaki, T. (1994) Involvement of a maternally transcribed lectin gene in the early development of Bombyx mori. Roux's Arch. Dev. Biol. 203, 397-401.

Suzuki, Y. (1994) Genes that are involved in Bombyx body plan and silk gene regulation. Int. J. Dev. Biol. 38, 231-235.

Xu, P.-X., Fukuta, M., Takiya, S., Matsuno, K., Xu, X. and Suzuki, Y. (1994) Promoter of the POU-M1/SGF-3 gene involved in the expression of Bombyx silk genes. J. Biol. Chem. 269, 2733-2742.

Xu, X., Xu, P.-X. and Suzuki, Y. (1994) . A maternal homeobox gene, Bombyx caudal, forms both mRNA and protein concentration gradients spanning anteroposterior axis during gastrulation. Develop. 120, 277-285.