Regulation of genes for ubiquitination and SUMO-specific protease involved in larval development of the silkworm, Bombyx mori.

Protein modifications with highly conserved small proteins, such as ubiquitin (Ub) and small ubiquitin-like modifier (SUMO), regulate various cellular processes; however, the contribution of these protein modifications to larval development in insects has not yet been elucidated. We investigated the regulation of genes for these protein modifications in the posterior silk gland (PSG) during larval development of the silkworm Bombyx mori. We found that several genes encoding enzymes (E1, E2, and E3) for ubiquitination and SUMO-specific protease were upregulated by 20-hydroxyecdysone (20E), and, consistently, increases in ubiquitinated proteins were observed during the fourth molting stage. An injection of 20E into larvae at the fourth feeding stage induced higher expression levels of these E1, E2, and E3 genes and ecdysis approximately one day earlier than in mock-treated larvae. The expression of the fibroin heavy-chain gene (fibH) was simultaneously suppressed approximately one day earlier in 20E-injected larvae. The treatment of cultured PSG with 20E also induced these genes, which could be categorized into at least two types: those induced by a high dose of 20E, or by a pulse of 20E. In contrast to the 20E treatment, the administration of PR-619, an inhibitor of Ub- and SUMO-specific proteases in larvae, delayed ecdysis and prolonged the expression of fibH. These results suggest that the regulation of genes for ubiquitination and SUMO-specific protease is involved in the larval development of B. mori.

In vivo fluorescence correlation spectroscopy analyses of FMBP-1, a silkworm transcription factor.

Fibroin modulator-binding protein 1 (FMBP-1) is a silkworm transcription factor that has a unique DNA-binding domain called the one score and three amino acid peptide repeat (STPR). Here we used fluorescence correlation spectroscopy (FCS) to analyze the diffusion properties of an enhanced green fluorescent protein-tagged FMBP-1 protein (EGFP-FMBP-1) expressed in posterior silk gland (PSG) cells of Bombyx mori at the same developmental stage as natural FMBP-1 expression. EGFP-FMBP-1 clearly localized to cell nuclei. From the FCS analyses, we identified an immobile DNA-bound component and three discernible diffusion components. We also used FCS to observe the movements of wild-type and mutant EGFP-FMBP-1 proteins in HeLa cells, a simpler experimental system. Based on previous in vitro observation, we also introduced a single amino acid substitution in order to suppress stable FMBP-1-DNA binding; specifically, we replaced the ninth Arg in the third repeat within the STPR domain with Ala. This mutation completely disrupted the slowest diffusion component as well as the immobile component. The diffusion properties of other FMBP-1 mutants (e.g. mutants with N-terminal or C-terminal truncations) were also analyzed. Based on our observations, we suggest that the four identifiable movements might correspond to four distinct FMBP-1 states: (a) diffusion of free protein, (b) and

A Hox Gene, Antennapedia, Regulates Expression of Multiple Major Silk Protein Genes in the Silkworm Bombyx mori.

Hoxgenes play a pivotal role in the determination of anteroposterior axis specificity during bilaterian animal development. They do so by acting as a master control and regulating the expression of genes important for development. Recently, however, we showed that Hoxgenes can also function in terminally differentiated tissue of the lepidopteranBombyx mori In this species,Antennapedia(Antp) regulates expression of sericin-1, a major silk protein gene, in the silk gland. Here, we investigated whether Antpcan regulate expression of multiple genes in this tissue. By means of proteomic, RT-PCR, and in situ hybridization analyses, we demonstrate that misexpression of Antpin the posterior silk gland induced ectopic expression of major silk protein genes such assericin-3,fhxh4, and fhxh5 These genes are normally expressed specifically in the middle silk gland as is Antp Therefore, the evidence strongly suggests that Antpactivates these silk protein genes in the middle silk gland. The putativesericin-1 activator complex (middle silk gland-intermolt-specific complex) can bind to the upstream regions of these genes, suggesting that Antpdirectly activates their expression. We also found that the pattern of gene expression was well conserved between B. moriand the wild species Bombyx mandarina, indicating that the gene regulation mechanism identified here is an evolutionarily conserved mechanism and not an artifact of the domestication of B. mori We suggest that Hoxgenes have a role as a master control in terminally differentiated tissues, possibly acting as a primary regulator for a range of physiological processes.

Regulation of Silk Genes by Hox and Homeodomain Proteins in the Terminal Differentiated Silk Gland of the Silkworm Bombyx mori.

The silk gland of the silkworm Bombyx mori is a long tubular organ that is divided into several subparts along its anteroposterior (AP) axis. As a trait of terminal differentiation of the silk gland, several silk protein genes are expressed with unique regional specificities. Most of the Hox and some of the homeobox genes are also expressed in the differentiated silk gland with regional specificities. The expression patterns of Hox genes in the silk gland roughly correspond to those in embryogenesis showing "colinearity". The central Hox class protein Antennapedia (Antp) directly regulates the expression of several middle silk gland-specific silk genes, whereas the Lin-1/Isl-1/Mec3 (LIM)-homeodomain transcriptional factor Arrowhead (Awh) regulates the expression of posterior silk gland-specific genes for silk fiber proteins. We summarize our results and discuss the usefulness of the silk gland of Bombyx mori for analyzing the function of Hox genes. Further analyses of the regulatory mechanisms underlying the region-specific expression of silk genes will provide novel insights into the molecular bases for target-gene selection and regulation by Hox and homeodomain proteins.

LIM-homeodomain transcription factor Awh is a key component activating all three fibroin genes, fibH, fibL and fhx, in the silk gland of the silkworm, Bombyx mori.

In the silkworm Bombyx mori, three fibroin genes, fibroin-heavy-chain (fibH), fibroin-light-chain (fibL) and fibrohexamerin (fhx), are coexpressed only in the posterior silk gland (PSG) cells, while the sericin genes encoding silk glue proteins are expressed in the middle silk gland (MSG) cells. Silk gland factor-2 (SGF-2) is a PSG-specific activator complex of fibH, composed of a LIM-homeodomain protein, Awh, and its cofactors, Ldb and Lcaf. We investigated whether SGF-2 can activate other fibroin genes using transgenic silkworms. The genes for Ldb and Lcaf were expressed ubiquitously in various tissues, while the gene for Awh was expressed strictly specific in PSG of the wild type silkworms. Misexpression of Awh in transgenic silkworms induced ectopic expression of fibL and fhx as well as fibH in MSG. Coincidently with the induction of fibL and fhx by Awh, binding of SGF-2 to the promoter of fibL and fhx was detected in vitro, and SGF-2 binds directly to the fhx core promoter. Ectopic expression of the fibroin genes was observed at high levels in the middle part of MSG. Moreover, fibL and fhx were induced in the anterior silk gland (ASG) of the transgenic silkworms, but fibH was not. These results indicate that Awh is a key activator of all three fibroin genes, and the activity is probably regulated in conjunction with additional factors.

Hox transcription factor Antp regulates sericin-1 gene expression in the terminal differentiated silk gland of Bombyx mori.

Hox genes are well-known master regulators in developmental morphogenesis along the anteroposterior axis of animals. However, the molecular mechanisms by which Hox proteins regulate their target genes and determine cell fates are not fully understood. The silk gland of Bombyx mori is a tubular tissue divided into several subparts along the anteroposterior axis, and the silk genes are expressed with specific patterns. The sericin-1 gene (ser1) is expressed in the middle silk gland (MSG) with sublocal specificity. Here we show that the Hox protein Antp is a component of the middle silk gland-specific complex, MIC (MSG-intermolt-specific complex), binds to the essential promoter element of ser1, and activates its expression. Ectopic expression of Antp in transgenic silkworms induced the expression of ser1 in the posterior silk gland (PSG), but not in the anterior part of MSG (MSG-A). Correspondingly, a MIC-like complex was formed by the addition of recombinant Antp in extracts from PSG with its cofactors Exd and Hth, but not in extracts from MSG-A. Splicing patterns of ser1 mRNA induced by the ectopic expression of Antp in PSG were almost the same as those in MSG at the fifth instar and altered depending on the induction timing of Antp. Other Hox genes were expressed with sublocal specificity in the silk gland. The Bombyx silk gland might provide a useful system for understanding how Hox proteins select and regulate their target genes.

Silk gland factor-2, involved in fibroin gene transcription, consists of LIM homeodomain, LIM-interacting, and single-stranded DNA-binding proteins.

SGF-2 binds to promoter elements governing posterior silk gland-specific expression of the fibroin gene in Bombyx mori. We purified SGF-2 and showed that SGF-2 contains at least four gene products: the silkworm orthologues of LIM homeodomain protein Awh, LIM domain-binding protein (Ldb), a sequence-specific single-stranded DNA-binding protein (Lcaf), and the silk protein P25/fibrohexamerin (fhx). Using co-expression of these factors in Sf9 cells, Awh, Ldb, and Lcaf proteins were co-purified as a ternary complex that bound to the enhancer sequence in vitro. Lcaf interacts with Ldb as well as Awh through the conserved regions to mediate transcriptional activation in yeast. Misexpression of Awh in transgenic silkworms induces ectopic expression of the fibroin gene in the middle silk glands, where Ldb and Lcaf are expressed. Taken together, this study demonstrates that SGF-2 is a multisubunit activator complex containing Awh. Moreover, our results suggest that the Ldb·Lcaf protein complex serves as a scaffold to facilitate communication between transcriptional control elements.

Inhibition of the binding of MSG-intermolt-specific complex, MIC, to the sericin-1 gene promoter and sericin-1 gene expression by POU-M1/SGF-3.

The sericin-1 gene encoding a glue protein is expressed in the middle silk gland (MSG) of the silkworm, Bombyx mori. A member of the class III POU domain transcription factors, POU-M1, was cloned as the factor bound to the SC site of the sericin-1 promoter and has been proposed to be a positive transcription factor. In this study, we analyzed the expression pattern of the POU-M1 gene in fourth and fifth instars in comparison with the pattern of the sericin-1 gene. The POU-M1 gene was expressed strongly in the region anterior to the sericin-1-expressing portion of the silk gland at both feeding stages. As the sericin-1-expressing region expands from the posterior to middle portions of the MSG in the fifth instar, the POU-M1-expressing region retreated from the middle to anterior portion. Introduction of the expression vector of POU-M1 into the silk glands by gene gun technology repressed promoter activity of the sericin-1 gene, suggesting that POU-M1 regulates the sericin-1 gene negatively. An in vitro binding assay showed that POU-M1 bound not only to the SC site but also to other promoter elements newly detected in vivo. Another spatiotemporal specific factor MIC binds to these elements, and POU-M1 competed with MIC to bind at the -70 site essential for promoter activity. These results suggest that POU-M1 is involved in restricting the anterior boundary of the sericin-1-expressing region in the silk gland by inhibiting the binding of the transcriptional activator to the promoter elements.

Novel enhancer and promoter elements indispensable for the tissue-specific expression of the sericin-1 gene of the silkworm Bombyx mori.

Sericins are glue proteins produced specifically in the middle silk gland (MSG) of the silkworm Bombyx mori, while the silk fiber protein, fibroin, is produced in the posterior silk gland (PSG). These silk proteins are expected to be useful biomaterials in medical technology as well as biotechnology. In this study, we analyzed promoter elements of the sericin-1 gene (ser1) in vivo by introducing reporter constructs into silk glands via gene gun technology. The region from -1602 to +47 was sufficient to induce MSG-specific expression. The 5' deletion mutants showed a three-step decrease in promoter activity with the key sequences located between -1362 and -1250, -201 and -116, and -115 and -37. We detected a tissue- and stage-specific factor complex (MSG-intermolt-specific complex: MIC) bound to the sequence elements around the -1350, -320, -180, and -70 regions. A mutation in the -70 region, which inhibits MIC-binding, diminished almost all promoter activity, while another mutation that did not inhibit MIC-binding showed no effect on promoter activity. The results suggest that the binding of MIC to the above elements is intrinsic for the spatiotemporal specificity of ser1 in vivo.

Protein kinase B gene homologue pkbR1 performs one of its roles at first finger stage of Dictyostelium.

Dictyostelium discoideum has protein kinases AKT/PKBA and PKBR1 that belong to the AGC family of kinases. The protein kinase B-related kinase (PKBR1) has been studied with emphasis on its role in chemotaxis, but its roles in late development remained obscure. The pkbR1 null mutant stays in the first finger stage for about 16 h or longer. Only a few aggregates continue to the migrating slug stage; however, the slugs immediately go back probably to the previous first finger stage and stay there for approximately 37 h. Finally, the mutant fingers diversify into various multicellular bodies. The expression of the pkbR1 finger protein probably is required for development to the slug stage and to express ecmB, which is first observed in migrating slugs. The mutant also showed no ST-lacZ expression, which is of the earliest step in differentiation to one of the stalk cell subtypes. The pkbR1 null mutant forms a small number of aberrant fruiting bodies, but in the presence of 10% of wild-type amoebae the mutant preferentially forms viable spores, driving the wild type to form nonviable stalk cells. These results suggest that the mutant has defects in a system that changes the physiological dynamics in the prestalk cell region of a finger. We suggest that the arrest of its development is due to the loss of the second wave of expression of a protein kinase A catalytic subunit gene (pkaC) only in the prestalk region of the pkbR1 null mutant.

STPR, a 23-amino acid tandem repeat domain, found in the human function-unknown protein ZNF821.

The STPR motif is composed of 23-amino acid repeats aligned contiguously. STPR was originally reported as the DNA-binding domain of the silkworm protein FMBP-1. ZNF821, the human protein that contains the STPR domain, is a zinc finger protein of unknown function. In this study, we prepared peptides of silkworm FMBP-1 STPR (sSTPR) and human ZNF821 STPR (hSTPR) and compared their DNA binding behaviors. This revealed that hSTPR, like sSTPR, is a double-stranded DNA-binding domain. Sequence-independent DNA binding affinities and α-helix-rich DNA-bound structures were comparable between the two STPRs, although the specific DNA sequence of hSTPR is still unclear. In addition, a subcellular expression experiment showed that the hSTPR domain is responsible for the nuclear localization of ZNF821. ZNF821 showed a much slower diffusion rate in the nucleus, suggesting the possibility of interaction with chromosomal DNA. STPR sequences are found in many proteins from vertebrates, insects, and nematodes. Some of the consensus amino acid residues would be responsible for DNA binding and concomitant increases in α-helix structure content.

DNA-binding property of the novel DNA-binding domain STPR in FMBP-1 of the silkworm Bombyx mori.

The STPR domain is a novel DNA-binding domain composed of repeats of 23 amino-acid-long peptide found in the fibroin-modulator-binding protein-1 (FMBP-1) of the silkworm Bombyx mori. Theoretical proteins having the STPR domain are highly conserved, particularly in vertebrates, but the functions are mostly unknown. In this study, the DNA-binding property of the STPR domain in FMBP-1 was examined. Use of reagents selecting the DNA groove and an oligonucleotide in which the dA:dT pairs of the probe were replaced with dI:dC pairs in mobility shift assay demonstrated that FMBP-1 approaches DNA from the major groove. Permutation electrophoresis using probes of the same length but containing the FMBP-1-binding site at different positions showed that FMBP-1 bends DNA through its binding. To induce the sharp bend of DNA, the STPR domain alone was insufficient and the long N-terminal extending region was necessary. Moreover, the basic region extending from the N-terminus of the STPR domain stabilized the DNA binding of the STPR domain. These results suggested that DNA-binding properties of the STPR domain are affected strongly by the structure of the flanking regions in the STPR domain-containing proteins.

Differential usage of two promoters of the Broad-Complex gene in the silkworm, Bombyx mori.

The Broad-Complex gene encodes one of the key regulators of the ecdysone signal cascade. We previously isolated part of the genomic DNA and cDNAs of Broad-Complex in Bombyx mori (BmBR-C). Here, we report structures of the entire genomic DNA and 5' untranslated region (5'-UTR) of the cDNAs. BmBR-C was found to span about 158 kbp including 13 exons. In the 5'-UTR, additional alternatively spliced exons were identified. The 5' ends of the cDNAs were mapped to two different positions, the distal promoter (P(dist)) and proximal promoter (P(prox)), separated by 86 kbp. Expression from these promoters was controlled differentially. Semi-quantitative PCR using cDNAs from the carcass, silk glands and fat body revealed that expression from P(prox) was changed moderately and expression from P(dist) was weak and constant during the fourth ecdysis. At the onset of pupation, expression from P(prox) was suppressed in all tissues, but that from P(dist) was induced in the carcass and ASG. In the fat body, expression from both promoters increased in the prepupal stage. A combination of promoters differing in responsiveness to an ecdysone signal may serve to achieve a complex regulation of downstream genes in reply to a simple hormonal signal.

Fibroin-modulator-binding protein-1 (FMBP-1) contains a novel DNA-binding domain, repeats of the score and three amino acid peptide (STP), conserved from Caenorhabditis elegans to humans.

The predicted transcriptional regulatory factor for the fibroin gene of the silkworm Bombyx mori, fibroin-modulator-binding protein-1 (FMBP-1), was purified by sequential DNA affinity column chromatography, and cDNA clones corresponding to FMBP-1 were isolated from a library. The N-terminal half of FMBP-1 has a weak similarity to the DNA-binding domain of several transcriptional regulatory factors in higher plants. The C-terminal half contains four tandem repeats of a novel 23 amino acid motif, which we named the score and three amino acid peptide (STP). Other genes containing STP repeats were found in Drosophila, Caenorhabditis elegans, mouse and human. Mutational analysis of FMBP-1 showed that the STP repeats form a novel DNA-binding domain. Sequences flanking STP repeats modulated DNA-binding activity. The FMBP-1 gene was expressed during the fourth to fifth instar. FMBP-1 activity appeared to be regulated at the transcriptional level and by the post-transcriptional modification.

Structure and expression of the gene encoding a Broad-Complex homolog in the silkworm, Bombyx mori.

The steroid hormone ecdysone (20-hydroxyexdysone) initiates metamorphosis and also larval ecdysis in many insects by activating a cascade of genes that includes primary response genes (early genes), most of which encode transcriptional regulators, and secondary response genes (late genes) regulated by the early genes. One of the early genes, Broad-Complex (BR-C), a key regulator of the ecdysone cascade, shares a common amino-terminal BTB domain which is fused by alternative splicing to one of four pairs of C(2)H(2) zinc finger domains (Z1, Z2, Z3, and Z4). cDNAs for BR-C (BmBR-C) were isolated from the silkworm Bombyx mori. These genes showed 90.3% and 98.2% amino acid identity with the Drosophila BR-C and Manduca BR-C in the N-terminal BTB domain; 96.0%, 90.7%, and 85.2% identity with the three zinc finger domains of the Drosophila Z1, Z2, and Z4 isoforms; and 96.3% and 98.1% identity with the two zinc finger domains of the Manduca Z2 and Z4 isoforms, respectively. Partial genomic sequencing (from the 3' region of the core sequence to the 3' region of the Z3 class zinc finger-coding sequence) of the BmBR-C gene showed that four exons coding the zinc finger domains are arranged the same as the BR-C gene in Drosophila. The amino acid sequence predicted from the genomic sequence corresponding to the BmBR-C Z3 class zinc finger domain is 100% identical to the Z3 isoforms of Drosophila and Manduca. We examined expression patterns of the BmBR-C isoforms during late larval to pupal development in the epidermis, fatbody and silk gland. During the metamorphic transformation, the epidermis and silk gland are completely histolyzed; however, the fat body survives into the adult phase. Expression patterns of BmBR-C during development differed extensively between the histolyzed group and the survival group. The BmBR-C expression patterns in silk glands also differed between the anterior and other areas (the middle and posterior silk glands).

Bombyx Y-box protein BYB facilitates specific DNA interaction of various DNA binding proteins independently of the cold shock domain.

A new member of the Y-box protein family of the silkworm Bombyx mori (BYB) was co-purified with the fibroin gene enhancer-binding protein FMBP-1, and stimulated the binding of FMBP-1 to its cognate DNA element. However, the stimulatory effect was not specific to FMBP-1, BYB also enhancing the binding of mammalian transcription factors OTF2, SP1 and AP2 to their specific binding elements. Besides the above transcription regulatory factors, BYB facilitated the binding of basal transcription factor TBP, and enhanced transcription from the adenovirus 2 major late promoter in a reconstituted transcription system. Moreover, BYB stimulated the reactions of some restriction endonucleases under cold conditions. The C-terminal region of BYB was sufficient for these stimulatory effects, and the highly conserved cold shock domain (CSD) in the N-terminal region was dispensable. GST-pull down experiments showed that the C-terminal region could interact with DNA independently of the CSD. The above results suggest that the C-terminal region of BYB causes the active interaction of various DNA binding proteins with their targets. Such a function of the C-terminal region of BYB may partly explain the functional diversity of Y-box proteins.

The DNA binding of insect Fork head factors is strongly influenced by the negative cooperation of neighbouring bases.

The Drosophila melanogaster Fork head and Bombyx mori SGF1/Fork head proteins are key regulators of tissue specific gene expression in the modified larval labial glands. Here we use the competitive electrophoretic mobility shift assay to create a detailed Fork head binding matrix and we investigate some unusual features of the Fork head interaction with DNA. We found that the Fork head-DNA interaction is context dependent--the binding specificity of the protein is partly determined by specific combinations of neighbouring bases. Although the total number of the sub-optimal dinucleotide steps is not high, the negative cooperation of neighbouring bases significantly contributes to the overall binding site specificity. Our results allow efficient recognition of insect Fork head binding sites and we show that the putative Fork head cognate elements preferentially accumulate in the near upstream region of genes abundantly expressed in the labial gland.

Role of the Core Promoter for the Preferential Transcription of Fibroin Gene in the Posterior Silk Gland Extract: (transcription/core promoter/fibroin gene/initiator/DNase I footprint).

Preferential transcription of the fibroin gene in homologous cell-free transcription systems prepared from the posterior silk gland (psg) of Bombyx mori is supported by the modulators upstream and downstream of the core promoter. However, we found that the cognate fibroin core promoter composed of the TATA box and transcription initiation region was indispensable for this preferential transcription in the psg extract. Studies on the transcriptional efficiencies of reconstituted fibroin promoters indicated that the fibroin core promoter functions synergistically with the modulators. The core promoter of adenovirus major late promoter (AdMLP) which carries the same TATA box sequence as the fibroin promoter could not substitute for the fibroin core promoter for the preferential transcription. In DNase I footprint experiments a characteristic protection pattern was obtained on the fibroin core promoter specifically in the psg extract but not in the HeLa cell extract, and the pattern on the fibroin core promoter was distinguished from that on AdMLP. Appearance of the characteristic footprint pattern on the core promoter and promoter activity of the fibroin gene were correlated in the psg extract. These results suggest that specific interaction of the transcription machinery with fibroin core promoter in the psg extract is a basis of psg specific transcription of the fibroin gene.

Differential Transcription of the Fibroin and Sericin-1 Genes in Cell-Free Extracts1 : (fibroin gene/sericin gene/differential transcription/nuclear extracts/Bombyx mori).

Devices of the extraction procedues and a recovery of the final supernatant as tiers accompanied with a transcription ability assessment of the individual tiers have enabled us to develop a variety of cell-free transcription systems. The latter step revealed that the factors necessary for faithful transcription as well as enhanced transcription form aggregates or complexes indicating an intrinsic nature of these factors to interact each other. Altogether 14 cell-free transcription systems have been developed from Bombyx mori embryos and tissues of various developmental stages and a cultured cell line, and screened for activities enhancing the basal promoter levels of the silk genes. Using these extracts a differential transcription of plural tissue-specific genes was tried. The fibroin gene was preferentially transcribed than the sericin-1 gene in the extracts from the posterior silk glands where the fibroin gene is specifically transcribed in vivo, while the sericin-1 gene was dominant to the fibroin gene in the extracts from the middle silk glands where the sericin-1 gene is specifically transcribed. Thus, these nuclear extracts offer us a biochemical clue assaying factors responsible for the differential transcription.

Fibroin Gene Transcription in the Embryonic Stages of the Silkworm, Bombyx mori: (fibroin gene/ faithful transcription/ embryonic stages/Bombyx mori).

Using a modified RNase mapping method the transcription of the fibroin gene in Bombyx mori embryos was analyzed. It is known that the silk gland development begins at stage 19 of the 30 embryonic stages and its morphological development completes by stage 25. RNA samples obtained from embryos of a Chinese strain C108 from stages 4 through 23 did not give a positive signal except a faint and transient transcript detected at stage 22. In RNA samples from later stage embryos of a Kanebo commercial strain Kin-Shu X Sho-Wa, a faint and ambiguous fibroin transcript was detected at stages 25 and 26, and a clear and accurate initiation of transcription of the gene was detected from stage 27 and increased greatly at stages 29 and 30 reaching a level of about 0.3 ng/embryo or about 1% of total RNA presumably in the posterior silk gland. These results indicate that the fibroin gene transcription begins for the first time after the completion of the embryonic silk gland development, and also suggest that around stages 25 to 27 are probably a critical time to search for the production and accumulation of a factor(s) governing the transcriptional regulation of the fibroin gene.