NATIONAL INSITUTE FOR BASIC BIOLOGY

National Institute for Basic Biology

Division of Cell Differentiation

Professor:
Yoshiaki Suzuki
Associate Professor:
Kohji Ueno
Research Associates:
Kaoru Ohno
Hiroki Kokubo
Graduate Students:
Katsuyoshi Matsunami (Graduate University for Advanced Studies)
Yoshinori Ueno (Graduate University for Advanced Studies)
Technical Staff:
Chikako Inoue


We conduct two well-associated projects. One is to understand how a special tissue like the silk gland of Bombyx mori differentiates along the developmental programs and results in transcribing a specific set of genes like the silk fibroin and sericin-1 genes. The other concerns with what the specificity of the Bombyx body plan is and how the developmental regulatory genes dictate 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 gene transcription

We have been trying to understand the networks of 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 provide an information about 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 and other regulatory genes, and identification of their target genes expressed in the labial segment, where the silk glands originate.

During the past year we have succeeded in purifying the SGF-2, a key transcription factor for the fibroin gene, which binds to FC, FD, and FE elements in the enhancer I and enhances transcription specifically in the posterior silk gland (K. Ohno et al., unpublished). The purification steps include SP-Sepharose FF, Source 30Q, Dyna beads-FE element multimers, BioSilect 250, Mini S, and Mini Q. The SGF-2 is a huge complex revealing an apparent molecular mass of about 1.1 MDa and accommodating 12 components; groups of p30/p32/p33, p36/p47B/p50B/p55, and p45/p46/p47G/p48/p50G. Through a partial peptide sequencing and PCR analysis, p36 was identified as a Lim homeodomain family protein, and p47B/p50B/p55 were as nuclear Lim interactor homologues. p30/p32/p33 were identified as variants of p25 which was known as an associating protein to the fibroin heavy chain-light chain complex. This finding raises the possibility that the p25 variants might be a chaperone to the transcription complex. By the use of antibodies against p45, p47B, p47G, and p50G, it was found that these components were not or barely detectable in the extract from the middle silk gland where the fibroin gene is not expressed. Functional reconstitution experiments as well as developmental expression pattern analysis on these components are being planned.

Previously we reported cloning and labial segment-specific expression of the Bombyx Scr. Silk gland specific transcription factor-1 (SGF-1) is known to interact with the SA site of the sericin-1 gene and FA and FB sites of the fibroin gene. Also previously, we reported the SGF-1 to be a new member of the fork head/HNF-3b family. Expression patterns of SGF-1/fkh mRNA and protein in developing embryos were also described. At stage 20, the transcripts and protein were detected in the invaginating silk glands. Interestingly, preceding the appearance of the Bombyx Fkh protein in the invaginating silk glands, Bombyx Scr disappeared from the spots. This observation suggests the possibility that the Bombyx Scr is necessary to determine the nature of the labial segment and induce the silk gland invagination accompanied by the Bombyx fkh expression but the Scr protein is probably not necessary for the direct induction of Bombyx fkh expression in the invagination spots (Kokubo et al., Dev. Biol., in press).

The following observations supported above suggestions. In the Nc/Nc embryos described by Itikawa in 1944 that lack the Bombyx Antp gene (Nagata et al., 1996), we observed ectopic expression of Bombyx Scr in the thoracic and abdominal segments. These ectopic expressions resulted in inducing ectopic formation of invaginating silk glands in the prothoracic, mesothoracic, and metathoracic segments all of which revealed ectopic expression of Bombyx Fkh (Kokubo et al., Dev. Biol., in press).

Fig. 1.
Expression of SGF-1/Bm Fkh (A) and SGF-3/POU-M1 (B) in embryonic silk gland. (A) At stage 23, signals are seen in the middle and posterior silk gland. Revised from Kokubo et al. (1996). (B) At stage 21, signals are weakened in the posterior region. Revised from Kokubo et al. (1997) Dev. Genes Evol. 206, in press. Arrowheads indicate the borders between the anterior, middle, and posterior silk gland. All scale bars represent 100 mm.


By the time when the blastokinesis finishes (stage 25) and the silk gland fully develops, the Bombyx fkh transcripts and protein were restricted to the middle and posterior regions of the silk gland (Fig. 1A). These results suggest that besides the role of transcription factor for the silk genes the Bombyx fkh/SGF-1 may play important roles during the silk gland development.

We reported that the POU-M1 which binds to the SC site of the sericin-1 gene accommodates a POU-domain identical to Drosophila Cf1-a. The expression of the POU-M1 gene has been analyzed in Bombyx embryos by in situ hybridization and immunohistochemistry (Fig. 2). The gene was expressed specifically for the first time at stage 18-19 in a pair of restricted sites of the labial segment where a pair of prothoracic glands is going to be formed by invagination. After the silk gland invagination, the POU-M1 expression was detected in the developing silk gland and confined to the anterior and middle portions of the silk gland by late embryonic stages (Fig. 1B).

A Bombyx homologue of trachealess has been cloned, identified by sequence comparison, and named Bm trh (K. Matsunami et al., unpublished). The Bm trh is expressed first at the invagination sites of trachea, and continued to be expressed along the trachea development. Later, it is also expressed in the invagination sites of the silk glands. The expression in the silk gland continues along the silk gland development but disappears first in the posterior silk gland and then in the middle silk gland leaving the expression only in the anterior silk gland. This expression pattern differs from that of trh in the Drosophila salivary gland.

Fig. 2.
Expression of SGF-3/POU-M1 in embryos. (A) At stage 20, signals are seen in precursor cells of the prothoracic glands (pg), adductor plates (adp), abductor plates (abp), silk glands (silg), subbuccal glands (subg), corpora allata (ca), tracheal system (tr), and in some cells of the central nervous system (CNS). (B) At stage 21, the salivary glands are elongated from the posterior part of the adp. Both invaginated cells in the anterior mandibular and posterior maxillary segments are fused to form the subg. (C) At stage 23, signals are also seen in the adp, abp, and the pg that reveal mature form. (D) Magnified in posterior region of an embryo at stage 21. Signals are seen in parts of the anus. (E) At stage 25, signals are seen in some cells of the brain (br) and the suboesophageal ganglion (sg), and in the ca (arrows). (F) At stage 25, signals are seen in the salivary glands (salg) and the adp. Anterior side is always to the left and dorsal side is up. All scale bars represent 100 mm. Revised from Kokubo et al. (1997) Dev. Genes Evol. 206, in press.


II. Genes associated with abdominal leg development

Through the studies on the mechanisms of abodominal leg development, we found that a high molecular weight protein (p260/270) was expressed specifically in abdominal leg cells (Fig. 3) during early embryonic stages and disappeared by a late stage. p260/270 consists of two polypeptides with molecular weights of 260 and 270 kDa. We have established a purification procedure for p260/270 and have raised an antibody against p260/270. Immunoblot analysis of the ECa/ECa (additional crescent) and EN/EN (new additional crescent) mutants (Itikawa, 1943), which lack the Bombyx abdominal-A gene (Ueno et al., 1992) and therefore do not express abdominal legs, demonstrated that both mutants lacked p260/270. Therefore we speculate the expression of p260/270 may be regulated by the Bombyx abdominal-A gene. cDNA cloning and sequencing demonstrated that p260 and 270 have structures similar to rat fatty acid synthase, which synthesizes palmitate. Most of the enzymatic domains for palmitate synthesis were well conserved in the amino acid sequences of p260 and p270, while the thioesterase domains of p260 and p270 were less conserved to that of rat fatty acid synthase (Ueno and Suzuki (1997), J. Biol. Chem. 272, 13519-13526). Purified p260/270 can transfer palmitate to cysteine residues of synthetic peptides in vitro. We propose that p260/270 may be involved in protein palmitoylation and may function in abdominal leg development.

Fig. 3.
Expression of p260/p270 in the embryonic abdominal regions at stage 21A. The scale bar represents 100 mm. Revised from Ueno and Suzuki (1997), J. Biol. Chem. 272, 13519-13526.


Publication List:
Kokubo, H., Takiya, S., Mach, V. and Suzuki, Y. (1996) Spatial and temporal expression pattern of Bombyx fork head/SGF-1 gene in embryogenesis. Dev. Genes Evol. 206, 80-85.
Mach, V., Ohno, K., Kokubo, H. and Suzuki, Y. (1996) The Drosophila Fork head factor directly controls larval salivary gland-specific expression of the glue protein gene Sgs3. Nucl. Acids Res. 24, 2387-2394.
Nagata, T., Suzuki, Y., Ueno, K., Kokubo, H., Xu, X., Hui, C.-c., Hara, W. and Fukuta, M. (1996) Developmental expression of the Bombyx Antennapedia homologue and homeotic changes in the Nc mutant. Genes to Cells 1, 555-568.

nibb-adm@nibb.ac.jp
Last Modified: 12:00, June 27, 1997