Early embryonic development of Bombyx.

Decades have passed since the early molecular embryogenesis of Drosophila melanogaster was outlined. During this period, the molecular mechanisms underlying early embryonic development in other insects, particularly the flour beetle, Tribolium castaneum, have been described in more detail. The information clearly demonstrated that Drosophila embryogenesis is not representative of other insects and has highly distinctive characteristics. At the same time, this new data has been gradually clarifying ancestral operating mechanisms. The silk moth, Bombyx mori, is a lepidopteran insect and, as a representative of the order, has many unique characteristics found in early embryonic development that have not been identified in other insect groups. Herein, some of these characteristics are introduced and discussed in the context of recent information obtained from other insects.

Immobilization of surface non-affinitive protein onto a metal surface by an external electric field.

We investigated an alternate technique to coat the surface with a protein having no surface affinity, without the use of any exotic chemical agents. An external electric field was utilized to prepare the protein coating on a metal substrate. Stainless steel (St) substrate and lysozyme (LSZ) were used as the surface to be coated and the model non-adsorptive protein, respectively. Dynamics of the adsorption of LSZ on the St surface in the presence and absence of an external electric potential (EEP) were monitored by in-situ ellipsometry. Applying negative surface potential (-0.4 V vs Ag/AgCl) forced the adsorption of LSZ onto the St surface where LSZ did not adsorb without applying any EEP. The repetition of the EEP-application and -cut-off indicated the controllability of the LSZ coating amount depending on the total duration of the EEP-application. The coated LSZ largely remained bound to the surface even by the cut-off of the external electric field, the ratio of which to the detached amount was roughly constant (approximately 7:3). Furthermore, the LSZ coated surface on the St substrate was found to be reversibly switched between being affinitive and non-affinitive to a typical model protein adsorbate (bovine serum albumin) by the EEP-application and cut-off.

Complexities in Bombyx germ cell formation process revealed by Bm-nosO (a Bombyx homolog of nanos) knockout.

Inheritance (sequestration of a localized determinant: germplasm) and zygotic induction are two modes of metazoan primordial germ cell (PGC) specification. vasa and nanos homologs are evolutionarily conserved germline marker genes that have been used to examine the ontogeny of germ cells in various animals. In the lepidopteran insect Bombyx mori, although the lack of vasa homolog (BmVLG) protein localization as well as microscopic observation suggested the lack of germplasm, classical embryo manipulation studies and the localization pattern of Bm-nosO (one of the four nanos genes in Bombyx) maternal mRNA in the egg raised the possibility that an inheritance mode is operating in Bombyx. Here, we generated Bm-nosO knockouts to examine whether the localized mRNA acts as a localized germ cell determinant. Contrary to our expectations, Bm-nosO knockout lines could be established. However, these lines frequently produced abnormal eggs, which failed to hatch, to various extent depending on the individuals. We also found that Bm-nosO positively regulated BmVLG expression at least during embryonic stage, directly or indirectly, indicating that these genes were on the same developmental pathway for germ cell formation in Bombyx. These results suggest that these conserved genes are concerned with stable germ cell production. On the other hand, from the aspect of BmVLG as a PGC marker, we showed that maternal Bm-nosO product(s) as well as early zygotic Bm-nosO activity were redundantly involved in PGC specification; elimination of both maternal and zygotic gene activities (as in knockout lines) resulted in the apparent lack of PGCs, indicating that an inheritance mechanism indeed operates in Bombyx. This, however, together with the fact that germ cells are produced at all in Bm-nosO knockout lines, also suggests the possibility that, in Bombyx, not only this inheritance mechanism but also an inductive mechanism acts in concert to form germ cells or that loss of early PGCs are compensated for by germline regeneration: mechanisms that could enable the evolution of preformation. Thus, Bombyx could serve as an important organism in understanding the evolution of germ cell formation mechanisms; transition between preformation and inductive modes.

A Bombyx homolog of ovo is a segmentation gene that acts downstream of Bm-wnt1(Bombyx wnt1 homolog).

Insect embryogenesis is divided into long and short/intermediate germ types. The long germ type may exhibit Drosophila-like hierarchical segmentation mechanisms, whereas the short/intermediate type assumes some repeating mechanisms that are considered to be ancestral. Embryogenesis in Bombyx mori possesses both characteristics. Here, Bombyx ovo homolog (Bm-ovo) was identified as a gene involved in segmentation. Ovo is a Drosophila gene that encodes a zinc finger transcription factor and studies on its homolog functions in other systems have suggested that it acts as a switch to enable the initiation of differentiation from a progenitor cell state. This is the first description for ovo homologs being involved in insect segmentation. Bm-ovo is expressed dynamically during embryogenesis in a pattern that resembles that of gap and pair-rule genes. In Bm-ovo RNAi knockdown embryos, posterior segmentation does not proceed. In addition, defects in anterior segments are observed. In Bm-wnt1 knockdown embryos, the Bm-ovo expression pattern was changed, suggesting that Bm-wnt1 is an upstream regulator of Bm-ovo. The involvement of Bm-ovo may represent a novel ancestral step under the control of wnt genes in insect segmentation: this step may resemble those operating in cell differentiation processes.

Hunchback knockdown induces supernumerary segment formation in Bombyx.

Insect segment number within species appears to be fixed irrespective of germ types: long vs. short/intermediate. The present study showed induction of supernumerary segment formation by the knockdown of Bombyx hunchback (Bm-hb), presumably by terminal segment addition, a short/intermediate-like-segmentation mode that is not observed in normal Bombyx embryogenesis. This suggests that Bm-hb suppresses segmentation. The results obtained also suggest that the gap gene Bm-Kr (Bombyx Krüppel) provides a permissive environment for the progression of segmentation by suppressing the expression Bm-hb, which terminates segmentation. This indicates a novel mechanism by which the gap gene is involved in segmentation. It appears that Bm-Kr and Bm-hb are involved in segment counting and their interplay contributes to the correct number of segments being formed in Bombyx. Similar mechanisms may be operating in insects that employ the non-Drosophilan mode of segmentation such as in short/intermediate-germ insects.

Knockout silkworms reveal a dispensable role for juvenile hormones in holometabolous life cycle.

Insect juvenile hormones (JHs) prevent precocious metamorphosis and allow larvae to undergo multiple rounds of status quo molts. However, the roles of JHs during the embryonic and very early larval stages have not been fully understood. We generated and characterized knockout silkworms (Bombyx mori) with null mutations in JH biosynthesis or JH receptor genes using genome-editing tools. We found that embryonic growth and morphogenesis are largely independent of JHs in Bombyx and that, even in the absence of JHs or JH signaling, pupal characters are not formed in first- or second-instar larvae, and precocious metamorphosis is induced after the second instar at the earliest. We also show by mosaic analysis that a pupal specifier gene broad, which is dramatically up-regulated in the late stage of the last larval instar, is essential for pupal commitment in the epidermis. Importantly, the mRNA expression level of broad, which is thought to be repressed by JHs, remained at very low basal levels during the early larval instars of JH-deficient or JH signaling-deficient knockouts. Therefore, our study suggests that the long-accepted paradigm that JHs maintain the juvenile status throughout larval life should be revised because the larval status can be maintained by a JH-independent mechanism in very early larval instars. We propose that the lack of competence for metamorphosis during the early larval stages may result from the absence of an unidentified broad-inducing factor, i.e., a competence factor.

Analyses of interactions among pair-rule genes and the gap gene Krüppel in Bombyx segmentation.

In the short-germ insect Tribolium, a pair-rule gene circuit consisting of the Tribolium homologs of even-skipped, runt, and odd-skipped (Tc-eve, Tc-run and Tc-odd, respectively) has been implicated in segment formation. To examine the application of the model to other taxa, I studied the expression and function of pair-rule genes in Bombyx mori, together with a Bombyx homolog of Krüppel (Bm-Kr), a known gap gene. Knockdown embryos of Bombyx homologs of eve, run and odd (Bm-eve, Bm-run and Bm-odd) exhibited asegmental phenotypes similar to those of Tribolium knockdowns. However, pair-rule gene interactions were similar to those of both Tribolium and Drosophila, which, different from Tribolium, shows a hierarchical segmentation mode. Additionally, the Bm-odd expression pattern shares characteristics with those of Drosophila pair-rule genes that receive upstream regulatory input. On the other hand, Bm-Kr knockdowns exhibited a large posterior segment deletion as observed in short-germ insects. However, a detailed analysis of these embryos indicated that Bm-Kr modulates expression of pair-rule genes like in Drosophila, although the mechanisms appear to be different. This suggested hierarchical interactions between Bm-Kr and pair-rule genes. Based on these results, I concluded that the pair-rule gene circuit model that describes Tribolium development is not applicable to Bombyx.

Primordial germ cells in an oligochaete annelid are specified according to the birth rank order in the mesodermal teloblast lineage.

The primordial germ cells (PGCs) in the oligochaete annelid Tubifex tubifex are descentants of the mesodermal (M) teloblast and are located in the two midbody segments X and XI in which they serve as germline precursors forming the testicular gonad and the ovarian gonad, respectively. During embryogenesis, vasa-expressing cells (termed presumptive PGCs or pre-PGCs) emerge in a variable set of midbody segments including the genital segments (X and XI); at the end of embryogenesis, pre-PGCs are confined to the genital segments, where they become PGCs in the juvenile. Here, using live imaging of pre-PGCs, we have demonstrated that during Tubifex embryogenesis, pre-PGCs (defined by Vasa expression) stay in segments where they have emerged, suggesting that it is unlikely that pre-PGCs move intersegmentally during embryogenesis. Thus, it is apparent that pre-PGCs derived from the 10th and 11th M teloblast-derived primary m blast cells (designated m10 and m11) that give rise, respectively, to segments X and XI are specified in situ as PGCs and that those born in other segments become undetectable at the end of embryogenesis. To address the mechanisms for this segment-specific development of PGCs, we have performed a set of cell-transplantation experiments as well as cell-ablation experiments. When m10 and m11 that are normally located in the mid region of the embryo were placed in positions near the anterior end of the host embryo, these cells formed two consecutive segments, which exhibited Vasa-positive PGC-like cells at early juvenile stage. This suggests that in terms of PGC generation, the fates of m10 and m11 remain unchanged even if they are placed in ectopic positions along the anteroposterior axis. Nor was the fate of m10 and m11 changed even if mesodermal blast cell chains preceding or succeeding m10 and m11 were absent. In a previous study, it was shown that PGC development in segments X and XI occurs normally in the absence of the overlying ectoderm. All this strongly suggests that irrespective of their surrounding cellular environments, m10 and m11 autonomously generate PGCs. We propose that m10 and m11 are exclusively specified as precursors of PGCs at the time of their birth from the M teloblast and that the M teloblast possesses a developmental program through which the sequence of mesodermal blast cell identities is determined.

Anterior and posterior centers jointly regulate Bombyx embryo body segmentation.

Insect embryo segmentation is largely divided into long and short germ types. In the long germ type, each segment primordium is represented on a large embryonic rudiment of the blastoderm, and segmental patterning occurs nearly simultaneously in the syncytium. In the short germ type, however, only anterior segments are represented in the small embryonic rudiment, usually located on the egg posterior, and the rest of the segments are added sequentially from the posterior growth zone in a cellular context. The long germ type is thought to have evolved from the short germ type. It is proposed that this transition, which appears to have occurred multiple times over the course of evolution, was realized through the acquisition of a localized anterior instruction center. Here, I examined the early segmentation process in the silkmoth Bombyx mori, a lepidopteran insect, in which the mechanisms of anterior-posterior (AP) axis formation have not been well analyzed. In this insect, both the long germ and short germ features have been reported. The mRNAs for two key genes involved in insect AP axis formation, orthodenticle (Bm-otd) and caudal (Bm-cad), are localized maternally in the germ anlage, where they act as anterior and posterior instruction centers, respectively. RNAi studies indicate that, while Bm-cad affects the formation of all the even skipped (Bm-eve) stripes, there is also anterior Bm-eve stripe formation activity that involves Bm-otd. Thus, there is redundancy in Bm-eve stripe formation activity that must be coordinated. Some genetic interactions, identified either experimentally or hypothetically, are also introduced, which might enable robust AP formation in this organism.

Characterization of Bombyx embryo segmentation process: expression profiles of engrailed, even-skipped, caudal, and wnt1/wingless homologues.

To gain insight into segmentation processes, the expression at embryonic stages of the silkmoth Bombyx mori homologues of even-skipped (eve), engrailed (en), caudal (cad), and wnt1/wingless (wg) transcripts were examined by whole mount in situ hybridization. Pair-rule eve stripes and segmental en and wnt1/wg stripes were generated sequentially from anterior to posterior, confirming the previous results that showed that Bombyx belongs to short-germ insects. However, unlike in previously described short germ insects, the segmentation of Bombyx occurred without marked germ band elongation: the putative growth zone was expanded compared with previously described short germ insects. This may indicate that Bombyx represents an evolutionarily intermediate state in a transition from short to long germ type. The expressions of cad and wnt1/wg, which are known to be present in the growth zone in short germ insects, initially showed a large median expression domain that, as segmentation proceeded, later retracted to the posterior pole. This is also unique to this insect. Detailed analysis of their relative expressions indicated that wnt1/wg domain retracted faster than the cad domain, and double stain in situ hybridization suggested that the eve stripe appears from cells that have ceased to express wnt1/wg. Another unique aspect of Bombyx embryogenesis is that gastrulation began at later embryonic stage compared with other insects and proceeded slowly from anterior to posterior. On the basis of these results, conserved and divergent aspects of the evolution of insect segmentation mechanisms and germ cell formation are discussed.

Germ cell specification and early embryonic patterning in Bombyx mori as revealed by nanos orthologues.

In the silkmoth Bombyx mori, the germ cells first appear from the posterior ventral side of the egg (from within the mesodermal primordium) after blastoderm formation. This is in contrast to Drosophila, where germ cells appear at the posterior pole before cellular blastoderm formation. To date, germ plasm has not been found in B. mori. In this study, we describe the identification and expression pattern of nanos from B. mori, in which we recovered four nanos orthologues. One orthologue showed strong expression in embryonic germ cells, which was traced back to periplasmic granules dispersed on the ventral midline of the egg from the posterior-ventral focus of preblastoderm embryos. This suggests that, in B. mori, as in dipterans, germ cell formation depends on a localized determinant in the egg. The expression of another orthologue was observed in the posterior of the germ band. We speculate that nanos has dual functions; one in germ cell formation and the other in posterior body patterning, which is conferred by one nanos gene in Drosophila, but is assigned to different genes in B. mori.

The uptake mechanism of gallium-67 into hepatocytes treated with carbon tetrachloride.

We have recently reported that transferrin (Tf)-unbound gallium-67 (67Ga) may be taken up into the liver of carbon tetrachloride (CCl4)-treated rats. In the present study, we attempted to clarify detailed mechanism of Tf-unbound 67Ga uptake by hepatocytes treated with CCl4 using in vitro experimental system. Hepatotoxic damages by CCl4 are mostly attributed to radical formed by an action of cytochrome P450. P450 isozymes have a higher expression in the perivenous hepatocytes (PVH) more than periportal hepatocytes (PPH). Therefore, we thought that the uptake of 67Ga which had been used for the detection of liver damage might have a zonal difference. The results of ALT activities showed that the CCl4 exposure for 4 h strongly impaired PVH more than PPH. The uptake of 67Ga by PVH treated with CCl4 was also higher than that by PPH. Moreover, the uptake of 45Ca by PVH was higher than that by PPH. In order to investigate whether 67Ga passed through calcium channel of hepatocytes, we made use of calcium channel blocker and activator. The Ca2+-channel blocker, verapamil, significantly decreased the uptakes of 45Ca and 67Ga by PPH and PVH pretreated with CCl4. The addition of the Ca2+-channel activator, Bay K8644, significantly increased the uptake both of 45Ca and 67Ga by PPH pretreated with CCl4. In the present study, it was demonstrated that the uptake of Tf-unbound 67Ga preferentially occurred in CCl4-damaged PVH and 67Ga was taken up into the hepatocytes in part through calcium channel.

Zonal differences in gallium-67 uptakes between perivenous versus periportal regions of rat liver following carbon tetrachloride treatment.

Gallium-67 ((67)Ga) has been used as a tumor or inflammation-imaging agent in nuclear medicine, although underlying mechanism has not been fully elucidated. To gain some insights into the mechanism of (67)Ga uptake by injured liver, we analyzed the difference between perivenous and periportal regions of rat liver in terms of (67)Ga uptake by hepatocytes at the site of inflammation caused by carbon tetrachloride (CCl(4))-treatment. Distribution of (67)Ga in rat liver sections was monitored with a BAS5000 system following hepatic injury by CCl(4)-treatment. Periportal hepatocytes (PPH) and perivenous hepatocytes (PVH) were prepared by modified digitonin-collagenase perfusion technique. Uptakes of (67)Ga in PVH region and PPH region reached to a maximum 2 days after CCl(4)-treatment, and the amount of maximum uptake of (67)Ga in PVH was twice as much as that in PPH. Liver damage as measured by lipid peroxidation and (45)Ca uptake occurred in PVH region within 1 day after CCl(4)-treatment. Incorporation of bromodeoxyuridine into hepatocytes reached to a maximum 2 days after CCl(4)-treatment, and peaked amount of DNA synthesis in PVH was twice as much as that in PPH. These results indicated that the uptake of (67)Ga by the PVH region was carried out during hepatic regeneration phase rather than hepatic damage period by CCl(4)-treatment.

Expression pattern of Bombyx vasa-like (BmVLG) protein and its implications in germ cell development.

Germ cell development in the silkworm Bombyx mori is interesting in that the species has no recognizable germ plasm, and its germ cells appear first on the ventral side of the embryo, not on the posterior pole as in Drosophila melanogaster. We previously reported the isolation of a vasa homologue (BmVLG) from B. mori and revealed the specific expression of transcript in the germ cells. In this paper, we describe the embryonic expression pattern of BmVLG protein. Consistent with the lack of recognizable germ plasm, the protein is not localized in freshly laid eggs, and its specific expression is first detectable several hours after energids penetrate the periplasm. This is in contrast to D. melanogaster, where germ cell lineage can be traced with anti-vasa antibody just after the formation of pole cells as they sequester vasa-positive germ (pole) plasm during cellularization. It is also revealed that, within the first few hours of their appearance when extensive cell movement does not seem to occur, stained cells are sometimes widely dispersed along the midline, which eventually may lead to the formation of ectopic germ cells. The implications of these results for germ cell development are discussed.