Mori Embryos Research Articles

Electroporation, an alternative to biolistics for transfection of Bombyx mori embryos and larval tissues

There are few powerful techniques available to transfect insect tissues. We previously used biolistics to transfect Bombyx mori embryos, and larval and pupal tissues (Thomas J-L et al. 2001. Journal of Insect Science 1/9, Kravariti L et al. 2001. Insect Biochemistry and Molecular Biology 31: 473–479). As the main limitation was the irregularity in results we explored electroporation as an alternative technique by adapting techniques used for chicken embryos to B. mori embryos. By injecting the DNA solution into the hemocoel of late embryos that were finishing organogenesis, we expressed marker genes in numerous tissues following electroporation. With some adaptation of the method this was also achieved for early embryos lacking a hemocoel. Some larval tissues were also transfected. During these technical studies we found that optimizing parameters such as electrical voltage, number of pulses and their frequency, and conductivity of the buffer was important. These results confirmed that electroporation is a reliable technique for transfecting B. mori tissues.

Electroporation, an alternative to biolistics for transfection of Bombyx mori embryos and larval tissues

There are few powerful techniques available to transfect insect tissues. We previously used biolistics to transfect Bombyx mori embryos, and larval and pupal tissues (Thomas J-L et al. 2001. Journal of Insect Science 1/9, Kravariti L et al. 2001. Insect Biochemistry and Molecular Biology 31: 473-479). As the main limitation was the irregularity in results we explored electroporation as an alternative technique by adapting techniques used for chicken embryos to B. mori embryos. By injecting the DNA solution into the hemocoel of late embryos that were finishing organogenesis, we expressed marker genes in numerous tissues following electroporation. With some adaptation of the method this was also achieved for early embryos lacking a hemocoel. Some larval tissues were also transfected. During these technical studies we found that optimizing parameters such as electrical voltage, number of pulses and their frequency, and conductivity of the buffer was important. These results confirmed that electroporation is a reliable technique for transfecting B. mori tissues.

A possible role of 20-hydroxyecdysone in embryonic development of the silkworm Bombyx mori.

It has been well established that eggs of insects, including those of the silkworm Bombyx mori, contain various ecdysteroids and the amounts of these ecdysteroids fluctuate during embryonic development. In order to know the function of egg ecdysteroids in embryonic development of B. mori, we examined the biological activities of various egg ecdysteroids by in vitro ligand-binding assay and bioassay using B. mori eggs. First, using the ecdysteroid receptor of B. mori (BmEcR-B1/BmUSP heterodimer) prepared by yeast and Escherichia coli expression systems, the interaction between the ecdysteroid receptor and various egg ecdysteroids of B. mori was analyzed. The relative binding affinities of egg ecdysteroids to the BmEcR-B1/BmUSP heterodimer decreased in the order of 20-hydroxyecdysone > 2-deoxy-20-hydroxyecdysone > 22-deoxy-20-hydroxyecdysone > ecdysone > 2-deoxyecdysone > ecdysone 22-phosphate. Next, several egg ecdysteroids of B. mori were injected into the prospective diapause eggs, which show a very low level of free ecdysteroids at the onset of embryonic diapause (gastrula stage). Approximately 7% of them (P < 0.002, chi(2)-test) developed beyond the gastrula stage without entering diapause by the injection of 20-hydroxyecdysone (25 ng/egg). In contrast, the injection of other ecdysteroids was not effective in inducing embryonic development. These results suggest that 20-hydroxyecdysone, via the ecdysteroid receptor, is responsible for the developmental difference between diapause and non-diapause in B. mori embryos. Furthermore, it was suggested that continuous supply of 20-hydroxyecdysone may be required to induce embryonic development.

A helium burst biolistic device adapted to penetrate fragile insect tissues

To compensate for the extremely low penetration efficiency of the original PDS/1000-He Bio Rad biolistic device and the deleterious blast effect, design modifications have been made to the launching module. These modifications were evaluated on Bombyx mori embryos and fragile tissues, such as oocytes and imaginal wing disks. The original floppy macrocarrier was replaced by a rigid macrocarrier to avoid the effects of the helium blast. The efficiency of the gene gun bombardment was reinforced by the addition of a focusing nozzle. The reduced blast effect allowed us to carry out high-pressure shootings to small organs with improved penetration. This system allowed potentially all the internal embryonic tissues to be transfected with optimal survival rates. The new module was effective on tissues that are difficult to transfect, such as the epithelial wing disk that is covered by a peripodial membrane, and the ovarian follicle cells that lie under the ovariole cell membrane. The new macrocarrier allowed both an aqueous delivery of particles and an ethanolic dry delivery. No significant differences were noted between these two modes of delivery. The major improvement is the possibility of high pressure shooting correlated with appreciable penetration and a weak blast effect.

Purification and characterization of an insect haemolymph protein promoting in vitro replication of the Bombyx mori nucleopolyhedrovirus.

We have identified a novel protein that promotes Bombyx mori nucleopolyhedrovirus (BmNPV) replication in vitro. This protein was purified from heat-treated haemolymph of B. mori larvae by gel filtration and ion exchange chromatography, and designated as promoting protein (PP). The molecular mass of native PP estimated by column chromatography and that of denatured PP estimated by SDS-PAGE were 9600 Da and 15200 Da, respectively, suggesting that native PP is composed of a single polypeptide and may behave in the column as if it is a smaller protein because of its conformation and/or adsorptive nature. Addition of the PP to the culture medium of SES-BoMo-15A cells derived from B. mori embryos resulted in the strong promotion of BmNPV replication. The promoting activity positively correlated with the amount of PP in the culture medium up to 1 microg/ml, above which maximum virus replication occurred and resulted in the highest budded virus production and polyhedrin promoter-mediated luciferase gene expression of 10000-fold and 6000-fold higher than those without PP, respectively. A cDNA encoding the PP precursor (prePP) was successfully cloned and sequenced. Comparison between the amino acid sequence deduced from the nucleotide sequence of prePP cDNA and the N-terminal 18 amino acids determined for the purified PP indicated that the prePP (154 amino acids) consisted of a mature PP polypeptide (136 amino acids) with a signal sequence at the N terminus. Recombinant PP expressed from the cDNA using a baculovirus vector was similar in molecular mass, immunoreactivity and promoting activity to the native PP.

Intracellular signaling mechanisms that mediates morphogenic actions of sonic hedgehog and bone morphogenetic proteins

A homologue of the segment polarity gene wnt-1 from Bombyx mori (Bmwnt-1) has been characterized. The segmentally reiterated pattern of Bmwnt-1 transcrip9t distribution in B. mori embryos suggested its segment polarity function. Maximal levels of Bmwnt-1 RNA during embryonic development were reached by stage 21A. In the larval stages, Bmwnt-1 was expressed in the fore- and hindwing discs, ovaries, testes and gut, reminiscent of the expression domains in Drosophila. Bmwnt-1 was expressed in the wing-margin area of both the fore- and hindwing discs. The pattern of wnt-1 expression in the hindwing discs was similar to that in the butterfly Precis coenia but subtle differences existed in forewing discs of the two species, which correlated well with the absence of proximal bands of pigmentation in the adult Bombyx wings. In addition, Bmwnt-1 was expressed in the silkglands and the expression was confined to the anterior sub-compartment within the middle silkglands throughout development from the embryonic to late larval stages. This domain of Bmwnt-1 expression overlapped with those of Cubitus interruptus (BmCi) and sericin-2 but excluded the Engrailed expression domain viz. the middle and posterior sub-compartments of middle silkglands. Bmwnt-1 expression was detected only during the intermoults and not in the moulting periods.

Morphogenesis in the Early Embryo of the Lepidopteran Bombyx mori

The regional patterns of nuclear and cell division and the changes in size and shape of the developing Bombyx mori embryo were monitored from just prior to blastoderm formation until the onset of gastrulation. Nuclear invasion of the periphery and subsequent cellularization occurs with a marked anterior to posterior gradient. Consequently, there is no syncytial blastoderm stage in which all nuclei simultaneously rest at the periphery. In addition, the posteriormost blastoderm cells do not form pole cells. Prior to gastrulation, the Bombyx blastoderm cells undergo several unusual types of cell behaviors, indicated in part by the modifications and specializations of their lateral edges. Subsequent to the completion of cellularization, localized regions of mitosis can be found within the germ anlage, but at the stage monitored, just prior to the onset of gastrulation, these regions do not appear to be the equivalent of the Drosophila mitotic domains. The implications of these findings for modeling how segmental fates are established in insects are discussed. This work confirms and extends the work of earlier histological investigations of Bombyx development.

A maternal homeobox gene, Bombyx caudal, forms both mRNA and protein concentration gradients spanning anteroposterior axis during gastrulation.

We have isolated a caudal (cad) homologue from a cDNA library of Bombyx mori embryos. The Bombyx cad cDNA encodes a protein of 244 amino acids. The homology between Drosophila and Bombyx homeodomains is 80%. Similar to Drosophila cad, there is no YPWM peptide sequence along the upstream of homeodomain. Northern blot hybridization with a Bombyx cad probe revealed the presence of single maternal transcript of 2.3 kb. A stronger signal of the transcripts was detected in unfertilized eggs and in eggs up to 36 hours after deposition. The transcripts decreased rapidly by 2 days and a weak signal was maintained until hatching. To analyse its spatial expression pattern, we have established a novel frozen sectioning method for in situ hybridization and immunohistochemistry experiments. The results showed that Bombyx cad transcripts accumulated first in the nurse cells and transferred into the oocyte at a defined time during oogenesis. The maternal transcripts of Bombyx cad formed a concentration gradient spanning the anteroposterior axis during the gastrulation stage and were restricted to the anal pad, the most posterior domain, after 2 days of embryogenesis; the Drosophila cad mRNA revealed the corresponding expression profile during the syncytial blastoderm stage. The Bombyx cad protein was not detected in the ovary and the first 9 hours of eggs, but was first detected evenly during cellular blastoderm stage. During gastrulation, Bombyx cad protein concentration gradients shifted along the anteroposterior axis coinciding with the shifting of the mRNA concentration gradients.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Changes in polysome content during development after diapause of Bombyx mori embryos

Undegraded polysomes could be extracted from eggs of Bombyx mori by cutting egg shells with a blade in a buffer containing high salt. The polysome content as measured by this method increased steeply during post-diapause development, which was commenced by long term chilling followed by hot HCl treatment of diapausing eggs. At the 24th hr of the post-diapause development the polysome content became 2.6-times the level at 0 h. The alteration of the polysome content paralleled that of the incorporation of [ 14C]leucine into acid-insoluble fractions investigated by modified Takami's embryonic culture system.