Mutation of a vitelline membrane protein, BmEP80, is responsible for the silkworm “Ming” lethal egg mutant

The egg stage is an important stage in the silkworm (Bombyx mori) life cycle. Normal silkworm eggs are usually short, elliptical, and laterally flattened, with a sometimes hollowed surface on the lateral side. However, the eggs laid by homozygous recessive “Ming” lethal egg mutants (l-e^m) lose water and become concaved around 1 h, ultimately exhibiting a triangular shape on the egg surfaces. We performed positional cloning, and narrowed down the region containing the gene responsible for the l-e^m mutant to 360 kb on chromosome 10 using 2287 F₂ individuals. Using expression analysis and RNA interference, the best l-e^m candidate gene was shown to be BmEP80. The results of the inverse polymerase chain reaction showed that an ~ 1.9 kb region from the 3′ untranslated region of BmVMP23 to the forepart of BmEP80 was replaced by a > 100 kb DNA fragment in the l-e^m mutant. Several eggs laid by the normal moths injected with BmEP80 small interfering RNAs were evidently depressed and exhibited a triangular shape on the surface. The phenotype exhibited was consistent with the eggs laid by the l-e^m mutant. Moreover, two-dimensional gel electrophoresis showed that the BmEP80 protein was expressed in the ovary from the 9th day of the pupa stage to eclosion in the wild-type silkworm, but was absent in the l-e^m mutant. These results indicate that BmEP80 is responsible for the l-e^m mutation.     

Gene expression profiling in the silkworm, Bombyx mori, during early embryonic development

We prepared a cDNA library for a microarray from eggs of the silkworm, Bombyx mori, at the germ-band formation (24 hours after fertilization) stage. Using a microarray constructed with 2,445 ESTs, we screened gene expression profiles during germ-band formation at six specific time points in the early embryonic stages (from the unfertilized egg to the formation of abdominal leg appendages), and determined 241 of these cDNAs to represent genes that were expressed differentially during the germ-band formation stage. These differentially expressed genes grouped into two clusters. In the early and late clusters, 203 and 38 genes were upregulated, respectively. In the upregulated clusters, we isolated several genes that were associated with development and cell communication, including egalitarian, RAD23b, innexin 2, and senescence-associated protein. Northern blot hybridization revealed that the expression patterns of 14 genes had changed in each of the stages. In this study, we assessed changes in the levels of gene expression in relation to the germ-band formation stages in whole Bombyx embryos.     

Two gap junction channel (innexin) genes of the Bombyx mori and their expression

Gap junctions are clusters of intercellular channels that are associated with embryonic development and neural signaling. Innexins, invertebrate gap junction proteins, have been identified in Drosophila and Caenorhabditis. Here, we report the isolation and characterization of two novel members of the insect innexin family, Bm inx2 and Bm inx4, from embryos of the silkworm, Bombyx mori, during the germ-band formation stage. Bm inx2 is a single copy gene with one exon, while Bm inx4 is a single copy gene with four exons and three introns. The predicted proteins show structural similarities with other innexin family members, including four transmembrane (TM) domains, two extracellular loops (ELs), one cytoplasmic loop (CL), and typical conserved amino acids. Bm inx2 is phylogenetically orthologous to the other insect inx2 genes, but Bm inx4 is not orthologous to any known innexin including Dm inx4. Interestingly, Northern blotting and in situ hybridization showed that Bm inx2 was variously expressed across all developmental stages and in various tissues, with high expression seen in the nervous system at the time of embryogenesis. In contrast, Bm inx4 was transiently expressed at the germ-band formation stage of embryogenesis, and was specifically expressed in the ovary and testis during the larval and pupal stages. The isolation and characterization of these novel genes should form the basis for further study of the functional events that occur during development and neuronal communication in B. mori.     

Developmental changes in the localization of protein kinase CK2 in non-diapause and diapause eggs of the silkworm, Bombyx mori

To analyze the role of protein kinase CK2 (CK2) during early embryogenesis in non-diapause and diapause of the silkworm, the distribution and localization of Bombyx mori CK2 (BmCK2) were investigated by an immunohistochemical technique using antibodies against the α- and β-subunits of BmCK2. Both were localized in blastoderm cells of non-diapause and diapause eggs until 24 h after oviposition. More than 24 h after oviposition, however, the distribution of BmCK2 was different in non-diapause and diapause eggs. In non-diapause eggs, BmCK2 was mainly localized in yolk cells. In contrast, in diapause eggs, the localization was mainly observed in germ-band cells. Furthermore, we confirmed that the RNA helicase-like protein that was localized together with BmCK2 in non-diapause eggs was phosphorylated by BmCK2 in vitro. These data suggest that the role of BmCK2 is different in non-diapause and diapause eggs.     

Serine protease P-IIc is responsible for the digestion of yolk proteins at the late stage of silkworm embryogenesis

In silkworms, yolk proteins comprise vitellin, egg-specific protein and 30K proteins, which are sequentially degraded by endogenous proteases strictly regulated during embryogenesis. Although the process has been extensively investigated, there is still a gap in the knowledge about the degradation of silkworm yolk proteins on the last two days of embryonic development. In the present study, we isolated and purified a gut serine protease P-IIc, which demonstrated optimal activity at 25 °C and pH 11. Semi-quantitative RT-PCR combined with western blotting showed that P-IIc was actively expressed and significantly accumulated in the gut on the last two days of embryogenesis. When natural yolk proteins were incubated with P-IIc in vitro, vitellin and ESP were selectively degraded. P-IIc also demonstrated activity towards 30K proteins as evidenced by rapid and complete digestion of BmLP1 and partial digestion of BmLP2 and BmLP3. Furthermore, RNAi knockdown of P-IIc in silkworm embryos significantly reduced the degradation rate of residual yolk proteins on embryonic day 10. Taken together, our results indicate that P-IIc represents an embryonic gut protease with a relatively broad substrate specificity, which plays an important role in the degradation of yolk proteins at the late stage of silkworm embryogenesis.     

Apolipophorin-III expression and low density lipophorin formation during embryonic development of the silkworm,Bombyx mori

We examined the expression of apolipophorin-III (apoLp-III) during embryonic development of the silkworm Bombyx mori. ApoLp-III mRNA was first expressed 24 h after oviposition, which corresponds to the time of germ band formation. The amount of apoLp-III in the eggs increased from day 2, peaked on day 4, and then gradually decreased until hatching (on day 9.5). ApoLp-III was apparently synthesized during early embryogenesis, as radioactive amino acids were incorporated into newly synthesized apoLp-III in three-day-old eggs. Moreover, radioactive apoLp-III was found only in the embryo and not in the extraembryonic tissue. KBr density gradient ultracentrifugation of egg homogenates showed that apoLp-III was associated with low-density lipophorin (LDLp). These results suggest that LDLp is required for the delivery of lipids for organogenesis during embryogenesis.     

Effects of egt gene transfer on the development of Bombyx mori

This study investigated the effects of gain of ecdysteroid UDP-glucosyltransferase (EGT) gene function mutation on the development of the silkworm, Bombyx mori. A novel piggyBac-derived plasmid containing the egt gene from B. mori nucleopolyhedrovirus (BmNPV) driven by a heat-shock protein (hsp) 23.7 promoter, with a neomycin-resistance gene (neo) controlled by the BmNPV ie-1 promoter and a green fluorescent protein gene (gfp) under the control of the B. mori actin 3 (A3) promoter was constructed. The vector was transferred into silkworm eggs by sperm-mediated gene transfer. Transgenic silkworms were produced after screening for neo and gfp genes and gene transfer was verified by polymerase chain reaction, dot-blot hybridization and western blotting. The hatching rate of G1 generation silkworm eggs was about 60% lower than that of normal silkworm eggs. The duration of the G1 generation larval period was extended, and the G2 generation pupal stage lasted four days longer than that in non-transgenic silkworms. The ecdysone blood level in G2 silkworms in the third instar molting stage was reduced by up to 90%. These results show that EGT suppressed transgenic silkworm molting, and that egt expression in egt-transgenic silkworms resulted in arrest of metamorphosis from pupae to moths.     

Xenopus MBD3 plays a crucial role in an early stage of development

DNA methylation plays a crucial role in gene silencing via recruitment of the proteins that specifically recognize methyl-CpG. In the present study, we have shown that two splicing isoforms of MBD3, xMBD3 and xMBD3LF, are the major methyl-CpG binding proteins in Xenopus eggs and early stage embryos. They were highly expressed in the eyes and central nerve system of tadpoles. Inhibition of the expression of xMBD3 by antisense oligonucleotides severely affected embryogenesis. Low-dose injection of antisense oligonucleotides specifically affected eye formation. An identical phenotype was observed on the forced expression of xMBD3 mutated in the methyl-CpG binding domain (MBD) and xMBD3LF, those of which lack methylated DNA binding activity. On the other hand, the eye-defective phenotype was not induced on the injection of truncated forms of mutant xMBD3 or xMBD3LF that contained MBD. We propose that MBD3, distinct from the case in mouse, plays a crucial role in the recognition of methylated genes as an intrinsic component of the complex to guide the corepressor complex during an early stage of Xenopus embryogenesis.     

Disappearance of chorion proteins from Bombyx mori eggs treated with HCl solution to prevent diapause

Bombyx mori eggs enter diapause immediately after completion of mesoderm segregation. HCl treatment of approximately 24-hour-old eggs (germband formation stage) is well known to be the most effective procedure to prevent entry into diapause, although the molecular mechanism remains unclear. In this study, we examined the protein composition of diapausing and nondiapausing eggs after various HCl treatments known to prevent or break diapause and found that proteins of approximately 11 and 8 kDa disappeared immediately after HCl treatment. Partial amino acid sequences of these proteins indicated that they were members of the chorion class A protein L12 family synthesized in follicle cells. Under the hypothesis that the chorion provides a barrier to oxygen, dechorionation of diapausing eggs induces resumption of embryonic development. Hence, to test this and other hypotheses about the function of these proteins, we used 20% SDS-PAGE with Coomassie Brilliant Blue staining to trace their disappearance from embryos and eggshells after treatment with HCl under different conditions and on polyvoltine, univoltine, and bivoltine silkworm races. Even when 10-day-old diapausing eggs were treated with HCl, which did not break diapause, the 11 and 8 kDa proteins disappeared. Our results suggest that disappearance of these proteins is not directly associated with preventing entry into or breaking a diapause state. Nevertheless, our results cannot completely rule out the possibility that the 11 and 8 kDa proteins function to block permeability of O₂ during the period when HCl treatment is physiologically effective to prevent diapause so that after the diapause system is established within the egg, even removing the 11 and 8 kDa proteins may not affect to prevent diapause. We also discuss the role of these proteins in choriogenesis.     

Differentially expressed genes in the ovary of the sixth day of pupal “Ming” lethal egg mutant of silkworm, Bombyx mori

The “Ming” lethal egg mutant (l-e^m) is a vitelline membrane mutant in silkworm, Bombyx mori. The eggs laid by the l-e^m mutant lose water, ultimately causing death within an hour. Previous studies have shown that the deletion of BmEP80 is responsible for the l-e^m mutation in silkworm, B. mori. In the current study, digital gene expression (DGE) was performed to investigate the difference of gene expression in ovaries between wild type and l-e^m mutant on the sixth day of the pupal stage to obtain a global view of gene expression profiles using the ovaries of three l-e^m mutants and three wild types. The results showed a total of 3,463,495 and 3,607,936 clean tags in the wild type and the l-e^m mutant libraries, respectively. Compared with those of wild type, 239 differentially expressed genes were detected in the l-e^m mutant, wherein 181 genes are up-regulated and 58 genes are down-regulated in the mutant strain. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis results showed that no pathway was significantly enriched and three pathways are tightly related to protein synthesis among the five leading pathways. Moreover, the expression profiles of eight important differentially expressed genes related to oogenesis changed. These results provide a comprehensive gene expression analysis of oogenesis and vitellogenesis in B. mori which facilitates understanding of both the specific molecular mechanism of the 1-e^m mutant and Lepidopteran oogenesis in general.     

Differential gene expression during early embryonic development in diapause and non-diapause eggs of multivoltine silkworm Bombyx mori

Quantification of the differential expression of metabolic enzyme and heat-shock protein genes (Hsp) during early embryogenesis in diapause and non-diapause eggs of the silkworm B. mori was carried out by semi-quantitative RT-PCR. Data analysis revealed that, the phosphofructokinase (PFK) expression started at a higher level in the early stage (6 h after oviposition) in non-diapause eggs, while in diapause induced eggs, it started at a lower level. However, the PFK gene expression in diapause eggs was comparatively higher than in non-diapause eggs. PFK facilitates use of carbohydrate reserves. The lower level of PFK gene expression in the early stage of diapause induced eggs but comparatively higher level of expression than in non-diapause eggs is due to enzyme inactivation via protein phosphorylation during early embryogenesis followed by de-phosphorylation in later stage. The sorbitol dehydrogenase-2 (SDH-2) gene was down regulated in diapause induced eggs up to 24 h and its expression levels in diapause induced eggs coincided with that of PFK gene at 48h in non-diapause eggs. During carbohydrate metabolism, there is an initial temporary accumulation of sorbitol which acts as protectant. The down regulation of SDH-2 gene during the first 24 hours in diapause induced eggs was due to the requirement of sorbitol as protectant. However, since the diapause process culminates by 48 h, the SDH-2 gene expression increased and coincided with that of PFK gene expression. The trehalase (Tre) gene expression was at a lower level in diapause induced eggs compared to non-diapausing eggs. The induction of Tre activity is to regulate uptake and use of sugar by the tissues. The non-diapause eggs revealed maximum expression of GPase gene with major fluctuations as well as an overall higher expression compared to diapause induced eggs. The diapause process requires less energy source which reflects lower activity of the gene. Heat shock protein (Hsp) genes (Hsp20.4, 40, 70, and 90) revealed differential levels of expression in both the eggs at all stages of embryonic development. The present study thus provides an overview of the differential expression levels of metabolic enzyme and Hsp genes in non-diapause and diapause induced eggs of multivoltine silkworm B. mori within 48 h after oviposition, confirming the major role of in early embryogenesis.     

Effect of metamorphosis on the major hemolymph proteins of the silkworm

During the metamorphosis of the silkworm, Bombyx mori, three major hemolymph proteins (MHPs) (molecular weights 17,000, 25,000, 27,000) were detected and found to be distributed in the hemolymph and in the tissues of several organs, such as the fat body, midgut, ovary, testis, and even eggs. The MHPs in eggs gradually decreased and disappeared during embryogenesis. The formation, distribution, and utilization of MHPs in tissues other than the gonad, however, were not affected by sex. Radioisotope experiments in vivo revealed that the MHPs were synthesized at an early period of the fifth larval instar. The synthesis of at least two of them occurred in the fat body. MHPs in the hemolymph entered the tissues at the onset of the larval-pupal transformation. On the basis of their appearance, distribution, and depletion, the MHPs may be classified as reserve proteins which are synthesized in the larval stage and utilized later in the developmental stages.     

Ecdysone Metabolism in Diapause Eggs and Non-Diapause Eggs of the Silkworm, Bombyx mori

In order to compare ecdysone metabolism between diapause eggs and non-diapause eggs of the silkworm, Bombyx mori, (3)H-ecdysone and its derivatives ((3)H-3-epiecdysone and (3)H-ecdysone 22-phosphate) were injected into the eggs at various stages during early embryogenesis, and the resultant labelled metabolites were analyzed by high-performance liquid chromatography. From the quantitative and qualitative changes in the labelled metabolites between diapause eggs and non-diapause eggs, it was demonstrated that epimerization of ecdysone occurred during early embryogenesis irrespective of the embryonic stage in both diapause eggs and non-diapause eggs, and that phosphorylation of ecdysone was a major metabolic step in diapause eggs, whereas dephosphorylation of ecdysone 22-phosphate and its subsequent hydroxylation at the C-20 and C-26 positions were characteristic in non-diapause eggs.     

Expression of metabolic enzyme genes and heat-shock protein genes during embryonic development in diapause and non-diapause egg of multivoltine silkworm <Emphasis Type="Italic">Bombyx mori</Emphasis>

The expression of metabolic enzyme genes and heat-shock protein genes (Hsp) during early embryogenesis in diapause and non-diapause eggs of the silkworm Bombyx mori was quantified by semi-quantitative RT-PCR. The trehalase gene (Tre) was expressed in non-diapause eggs up-to nine days, while in diapause eggs was not up regulated. The glycogen phosphorylase gene (GPase) was expressed in non-diapause eggs, whereas in diapause eggs a high level was observed in early stage, but down regulated in later stage. The phosphofructokinase gene (PFK) and sorbitol dehyrogenase-2 gene (SDH-2) expression was fluctuated in non-diapause eggs, whereas in diapause eggs these were expressed only at early stage and not observed in later stage. The glucose-6-phosphate dehydrogenase gene (G6P-DH) in non-diapause eggs was highly expressed during the differentiation phase and decreased in the organogenesis phase. In contrast to this, expression in diapause eggs was of low level during differentiation phase and of high level observed in the organogenesis phase. In the tissues, PFK and SDH-2 were selectively expressed in cuticle and midgut, whereas Tre expression was high in midgut and ovary of larvae incubated at 15°C. The Hsp (20.4, 20.8, 40, 70, and 90) were expressed in both diapause and non-diapause eggs. Their expression was, however, selective in tissues with Hsp20.4 in midgut and ovary, Hsp40 in head, Hsp70 in cuticle and Hsp90 in ovary and head in high amounts at 15°C. These results suggest that the metabolic enzyme genes studied except Hsp play a major role during embryogenesis of diapause and non-diapause silkworm.     

Purification and Functional Characterization of a Protein: Bombyx mori Human Growth Hormone Like Protein in Silkworm Pupa

Human growth hormone (hGH) is a peptide hormone secreted by eosinophils of the human anterior pituitary, and a regulatory factor for a variety of metabolic pathways. A 30-kD protein from the pupa stage of silkworm was detected by Western blotting and confirmed by immunoprecipitation based on its ability to bind to anti-hGH antibody. This protein, named BmhGH-like protein, was purified from fresh silkworm pupas through low-temperature homogenization, filtration, and centrifugation to remove large impurity particles. The supernatants were precipitated, resuspended, and passed through a molecular sieve. Further purification by affinity chromatography and two-dimensional electrophoresis resulted in pure protein for analysis by MS MALDI-TOF-MS analysis. An alignment with predicted proteins indicated that BmhGH-like protein consisted of two lipoproteins, which we named hGH-L1 and hGH-L2. These proteins belong to the β-trefoil superfamily, with β domains similar to the spatial structure of hGH. Assays with K562 cells demonstrated that these proteins could promote cell division in vitro. To further validate the growth-promoting effects, hGH-L2 was cloned from pupa cDNA to create recombinant silkworm baculovirus vBmNPV-hGH-L2, which was used to infect silkworm BmN cells at low titer. Flow cytometric analysis demonstrated that the protein shortened the G0/G1 phase of the cells, and enabled the cells to rapidly traverse the G1/S phase transition point to enter S phase and promote cell division. Discovery of hGH-like protein in silkworm will once again arouse people’s interest in the potential medicinal value of silkworm and establish the basis for the development of new hormone drugs.