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.     

The Drosophila innexin 7 gap junction protein is required for development of the embryonic nervous system

The Drosophila genome encodes eight members of the innexin family of gap junction proteins. Most of the family members are expressed in complex and overlapping expression patterns during Drosophila development. Functional studies and mutant analysis have been performed for only few of the innexin genes. The authors generated an antibody against Innexin7 and studied its expression and functional role in embryonic development by using transgenic RNA interference (RNAi) lines. The authors found Innexin7 protein expression in all embryonic epithelia from early to late stages of development, including in the developing epidermis and the gastrointestinal tract. In early embryonic stages, the authors observed a nuclear localization of Innexin7, whereas Innexin7 was found in a punctuate pattern in the cytoplasm and at the membrane of most epithelial tissues at later stages of development. During central nervous system (CNS) development, Innexin7 was expressed in cells of the neuroectoderm and the mesectoderm and at later stages of embryogenesis, its expression was largely restricted to a segmental pattern of few glia and neuronal cells derived from the midline precursors. Coimmunostaining experiments showed that Innexin7 is expressed in midline glia, and in two different neuronal cells, the pCC and MP2 neurons, which are pioneer cells for axon guidance. RNAi-mediated knock down was used to gain insight into the embryonic function of innexin7. Down-regulation of innexin7 expression resulted in a severe disruption of embryonic nervous system development. Longitudinal, posterior, and anterior commissures were disrupted and the outgrowth of axon fibers of the ventral nerve cord was aberrant, causing peripheral nervous system defects. The results suggest an essential role for innexin7 for axon guidance and embryonic nervous system development in Drosophila.     

Identification and developmental expression of the ets gene family in the sea urchin (Strongylocentrotus purpuratus)

A systematic search in the available scaffolds of the Strongylocentrotus purpuratus genome has revealed that this sea urchin has 11 members of the ets gene family. A phylogenetic analysis of these genes showed that almost all vertebrate ets subfamilies, with the exception of one, so far found only in mammals, are each represented by one orthologous sea urchin gene. The temporal and spatial expression of the identified ETS factors was also analyzed during embryogenesis. Five ets genes (Sp-Ets1/2, Sp-Tel, Sp-Pea, Sp-Ets4, Sp-Erf) are also maternally expressed. Three genes (Sp-Elk, Sp-Elf, Sp-Erf) are ubiquitously expressed during embryogenesis, while two others (Sp-Gabp, Sp-Pu.1) are not transcribed until late larval stages. Remarkably, five of the nine sea urchin ets genes expressed during embryogenesis are exclusively (Sp-Ets1/2, Sp-Erg, Sp-Ese) or additionally (Sp-Tel, Sp-Pea) expressed in mesenchyme cells and/or their progenitors. Functional analysis of Sp-Ets1/2 has previously demonstrated an essential role of this gene in the specification of the skeletogenic mesenchyme lineage. The dynamic, and in some cases overlapping and/or unique, developmental expression pattern of the latter five genes suggests a complex, non-redundant function for ETS factors in sea urchin mesenchyme formation and differentiation.     

Developmental expression and molecular characterization of two gap junction channel proteins expressed during embryogenesis in the grasshopper Schistocerca americana

Gap junctions are membrane channels that directly connect the cytoplasm of neighboring cells, allowing the exchange of ions and small molecules. Two analogous families of proteins, the connexins and innexins, are the channel-forming molecules in vertebrates and invertebrates, respectively. In order to study the role of gap junctions in the embryonic development of the nervous system, we searched for innexins in the grasshopper Schistocerca americana. Here we present the molecular cloning and sequence analysis of two novel innexins, G-Inx(1) and G-Inx(2), expressed during grasshopper embryonic development. The analysis of G-Inx(1) and G-Inx(2) proteins suggests they bear four transmembrane domains, which show strong conservation in members of the innexin family. The study of the phylogenetic relationships between members of the innexin family and the new grasshopper proteins suggests that G-Inx(1) is orthologous to the Drosophila 1(1)-ogre. However, G-Inx(2) seems to be a member of a new group of insect innexins. We used in situ hybridization with the G-Inx(1) and G-Inx(2) cDNA clones, and two polyclonal sera raised against different regions of G-Inx(1) to study the mRNA and protein expression patterns and the subcellular localization of the grasshopper innexins. G-Inx(1) is primarily expressed in the embryonic nervous system, in neural precursors and glial cells. In addition, a restricted stripe of epithelial cells in the developing limb, involved in the guidance of sensory growth cones, expresses G-Inx(1). G-Inx(2) expression is more widespread in the grasshopper embryo, but a restricted expression is found in a subset of neural precursors. The generally different but partially overlapping expression patterns of G-Inx(1) and G-Inx(2) supports the combinatorial character of gap junction formation in invertebrates, an essential property to generate specificity in this form of cell-cell communication.     

Innexin2 gap junctions in somatic support cells are required for cyst formation and for egg chamber formation in Drosophila

Germ cells require intimate associations with surrounding somatic cells during gametogenesis. During oogenesis, gap junctions mediate communication between germ cells and somatic support cells. However, the molecular mechanisms by which gap junctions regulate the developmental processes during oogenesis are poorly understood. We have identified a female sterile allele of innexin2 (inx2), which encodes a gap junction protein in Drosophila. In females bearing this inx2 allele, cyst formation and egg chamber formation are impaired. In wild-type germaria, Inx2 is strongly expressed in escort cells and follicle cells, both of which make close contact with germline cells. We show that inx2 function in germarial somatic cells is required for the survival of early germ cells and promotes cyst formation, probably downstream of EGFR pathway, and that inx2 function in follicle cells promotes egg chamber formation through the regulation of DE-cadherin and Bazooka (Baz) at the boundary between germ cells and follicle cells. Furthermore, genetic experiments demonstrate that inx2 interacts with the zero population growth (zpg) gene, which encodes a germline-specific gap junction protein. These results indicate a multifunctional role for Inx2 gap junctions in somatic support cells in the regulation of early germ cell survival, cyst formation and egg chamber formation. Inx2 gap junctions may mediate the transfer of nutrients and signal molecules between germ cells and somatic support cells, as well as play a role in the regulation of cell adhesion.     

Silkworm egg proteins at the germ-band formation stage and a functional analysis of BmEP80 protein

The patterning of embryos in early stages is a critical process for embryo development. In order to understand the molecular mechanism of early embryogenesis in silkworm, 2-DE combined with MALDI-TOF-MS technologies were used to analyze the proteins from diapause-destined eggs at the germ-band formation stage. From over 1000 spots, 93 were selected for analysis and data were obtained from 59 revealing 42 proteins. Gene Ontology annotation showed these proteins were involved in several biological processes at the germ-band formation stage, including cell stress response and protein folding, cell growth and migration, termination of diapause, and nutrition storage. Prominent among them was a new 80 kDa protein, named Bombyx mori egg protein 80 (BmEP80). BmEP80 was a component of the eggshell which was secreted by follicle cells during the late vitellogenesis stage to early choriogenesis stage (FCs −5 to +10). It disappears during early embryogenesis and RNAi against it resulted in the collapse of eggs, thus it is likely that BmEP80 is a new component of the silkworm vitelline membrane.     

Regulation of intermuscular electrical coupling by the Caenorhabditis elegans innexin inx-6

The innexins represent a highly conserved protein family, the members of which make up the structural components of gap junctions in invertebrates. We have isolated and characterized a Caenorhabditis elegans gene inx-6 that encodes a new member of the innexin family required for the electrical coupling of pharyngeal muscles. inx-6(rr5) mutants complete embryogenesis without detectable abnormalities at restrictive temperature but fail to initiate postembryonic development after hatching. inx-6 is expressed in the pharynx at all larval stages, and an INX-6::GFP fusion protein showed a punctate expression pattern characteristic of gap junction proteins localized to plasma membrane plaques. Video recording and electropharyngeograms revealed that in inx-6(rr5) mutants the anterior pharyngeal (procorpus) muscles were electrically coupled to a lesser degree than the posterior metacorpus muscles, which caused a premature relaxation in the anterior pharynx and interfered with feeding. Dye-coupling experiments indicate that the gap junctions that link the procorpus to the metacorpus are functionally compromised in inx-6(rr5) mutants. We also show that another C. elegans innexin, EAT-5, can partially substitute for INX-6 function in vivo, underscoring their likely analogous function.     

Specific expression of GFPuv-beta1,3-N-acetylglucosaminyltransferase 2 fusion protein in fat body of Bombyx mori silkworm larvae using signal peptide

Bombyxin (bx) and prophenoloxidase-activating enzyme (ppae) signal peptides from Bombyx mori, their modified signal peptides, and synthetic signal peptides were investigated for the secretion of GFP(uv)-beta1,3-N-acetylglucosaminyltransferase 2 (GGT2) fusion protein in B. mori Bm5 cells and silkworm larvae using cysteine protease deficient B. mori multiple nucleopolyhedrovirus (BmMNPV-CP(-)) and its bacmid. The secretion efficiencies of all signal peptides were 15-30% in Bm5 cells and 24-30% in silkworm larvae, while that of the +16 signal peptide was 0% in Bm5 cells and 1% in silkworm larvae. The fusion protein that contained the +16 signal peptide was expressed specifically in the endoplasmic reticulum (ER) and in the fractions of cell precipitations. Ninety-four percent of total intracellular beta1,3-N-acetylglucosaminyltransferase (beta3GnT) activity was detected in cell precipitations following the 600, 8000, and 114,000g centrifugations. In the case of the +38 signal peptide, 60% of total intracellular activity was detected in the supernatant from the 114,000g spin, and only 1% was found in the precipitate. Our results suggest that the +16 signal peptide might be situated in the transmembrane region and not cleaved by signal peptidase in silkworm or B. mori cells. Therefore, the fusion protein connected to the +16 signal peptide stayed in the fat body of silkworm larvae with biological function, and was not secreted extracellularly.     

家蚕血细胞特异表达新基因Bm04862的鉴定及表达

通过家蚕组织芯片数据筛选得到家蚕血细胞特异表达基因Bm04862,并首次对该基因进行了克隆与鉴定。应用RACE技术获得该基因全长,并对其进行生物信息学分析。Bm4862基因开放阅读框819 bp,共编码273个氨基酸残基,预测其为跨膜蛋白;通过q RT-PCR技术对其时空表达情况进行分析;结果显示Bm04862基因在家蚕血细胞中特异高表达,并在4龄眠期和预蛹2 d时达到表达高峰;构建Bm04862真核表达载体,转染Sf9细胞分析其蛋白的亚细胞定位情况,结果表明其定位于细胞核膜和部分细胞质中。此外,用大肠杆菌刺激蚕体24 h后,Bm04862基因表达水平显著上调,表明大肠杆菌可以诱导该基因的表达,由此推测该基因可能参与家蚕的免疫应答。这为深入研究该基因在家蚕免疫反应中的功能提供了参考。     

Characterization and identification of the integrin family in silkworm, Bombyx mori

As an important economic insect, Bombyx mori is also a useful model organism for lepidopteran insect. Integrins are evolutionarily conserved from sponges to humans, and play vital roles in many physiological and pathological processes. To explore their diverse functions of integrins in insect, eleven integrins including six α and five β subunits were cloned and characterized from silkworm. Our results showed that integrins from silkworm own more family members compared to other invertebrates. Among those α subunits, integrins α1, α2, and the other four subunits belong to PS1, PS2, and PS3 groups, respectively. The β subunits mainly gather in the insect βν group except the β1 subunit which belongs to the insect β group. Expression profiles demonstrated that the integrins exhibited distinct patterns, but were mainly expressed in hemocytes. α1 and β2 subunits are the predominant ones either in the embryogenesis or larva stages. Interestingly, integrins were significantly up-regulated after stimulated by 20-hydroxyecdysone (20-E) in vivo. These results indicate that integrins perform diverse functions in hemocytes of silkworm. Overall, our results provide a new insight into the functional and evolutionary features of integrins.     

Molecular Characterization,Localization, and Distribution of Innexins in the Silkworm, <Emphasis Type="Italic">Bombyx mori</Emphasis>

Gap junctions that allow for a direct exchange of second messenger and ions are the most conserved cellular structures in multicellular organisms. We have isolated and characterized a Bombyx mori gene innexin3 that encodes a new member of the innexin family required for the early embryonic development. The BmINX3 mRNA was 1,814 nucleotide residues in length, and the deduced amino acid sequence of BmInx3 shared 74% similarity with Apis melifera innexin3. The expression profile of the BmINX3 mRNA is similar to that of previously described BmINX2, expressed in ovary and testis after 5th instar larvae and in fat body after gut purge. However, during embryogenesis, the expression of BmINX3 mRNA is restricted to the blastokinesis stage. Microscopic observation of the BmInx2 and BmInx3 fused to fluorescent proteins showed an overlapping cytoplasmic expression, whereas the BmInx4 is accumulated in the cytoplasmic surface at which two cells have physical contact. This finding of innexins distribution in silkworm would provide an essential basis for future studies of the functions and interactions of innexins.     

Investigation of reduced serum and serum-free media for the cultivation of insect cells (Bm5) and the production of baculovirus (BmNPV)

An experimental study has been carried out to investigate the effectivenes of several reduced serum and serum-free media for the cultivation of an ovarian cell line, Bm5, of the lepidopteran insect Bombyx mori. Bm5 cell were successfully adapted to grow in a medium containing 5% serum and a serum-free medium (EX-CELL 400). On the other hand, this cell line could not be adapted to grow in several other media suggested in the literature, including IPL-41 + 2% fetal bovine serum (FBS), SF-900, and a serum-free medium (ISFM). Furthermore, a comparative study was conducted to determine the production levels of B. mori nuclear polyhedrosis virus (BmNPV) in Bm5 cells cultured in three different medium formulations. The production levels of BmNPV in adapted Bm5 cells grown in a 5% serum-supplemented medium and a serum-free medium (EX-CELL 400) were comparable to those obtained in Bm5 cells grown 10% serum-supplemented medium. (c) 1992 John Wiley & Sons, Inc.     

30K蛋白对家蚕细胞及幼虫存活的作用

利用杆状病毒表达系统在家蚕BombyxmoriL.细胞BmN中表达家蚕30K蛋白,以亲本病毒BmPAK6为对照,将重组病毒Bm/r-30K分别感染BmN细胞及家蚕幼虫,观察其感染后不同时间的凋亡及存活率。与对照相比,重组病毒感染后的BmN细胞的存活率明显高于对照组,并且感染的家蚕幼虫存活时间也较长,表明体内过量表达家蚕30K蛋白有助于延长其细胞及幼虫的存活。