Commencement of pupal commitment in late penultimate instar and its hormonal control in wing imaginal discs of the silkworm, Bombyx mori

Pupal commitment of the wing imaginal disc of the silkworm, Bombyx mori, is completed shortly after the final (fifth) larval ecdysis. Pupal commitment was induced by in vitro culture with 20-hydroxyecdysone (20E). Shortly after the head capsule slippage (HCS) that occurs approximately 24 h before the final larval ecdysis, the discs become competent to respond to 20E, indicating that the process of pupal commitment begins in the late penultimate (fourth) instar. The simultaneous presence of methoprene (JHA) with 20E suppressed the pupal commitment at 4 ng/ml for the discs at 12 h after HCS and at 240 ng/ml for the discs at the ecdysis. Thus, the discs rapidly lose their sensitivity to JH at the end of the fourth instar. Day 0 fourth wing discs were not pupally committed by 20E when freshly dissected discs were exposed to 20E. By contrast, exposure to 20E after a pre-culture in a hormone free medium induced the pupal commitment. In those discs, the effective JHA concentration to suppress the 20E effects was 0.1 ng/ml. The present data suggest that pupal commitment proceeds through two stages from a reversible state that begins at around HCS to an irreversible state early in the fifth instar. The loss of sensitivity to JH is the primary impetus to begin the process and 20E is the factor that drives the discs to enter the reversible state.     

Developmental profiles of wing imaginal discs of flügellos(fl), a wingless mutant of the silkworm, Bombyx mori

 Mutations at the flügellos (fl) locus in Bombyx mori give rise to wingless pupae and moths. To understand the developmental steps responsible for the fl wing defect, we compared the morphological changes and protein synthesis profiles between fl and wild-type (WT) wing discs during larval development. Morphologically, the four wing discs in the fl homozygote larva developed normally at least until the fourth instar, but they were slightly smaller than those of the WT. After the last larval ecdysis, wing epithelial invagination and tracheal migration into the lacunar spaces evidently occurred in the WT wing discs. However, there was no apparent morphological change in fl discs through the fifth instar. The fl wing discs cultured in medium containing 20-hydroxyecdysone (20E) did not grow and develop, although the WT wing discs extended and differentiated under the same conditions. A comparison of protein synthesis in the wing discs revealed that several bands were differentially expressed between the fl and WT. A 41-kDa band expressed abundantly from larval to pharate pupal stages in the WT wing discs was rarely observed in fl discs. Furthermore, in vitro culture studies showed that the 41-kDa protein was induced by 20E and specifically synthesized in WT wing discs after the wandering stage, but not in fl discs. The wing-specific protein synthesis and morphogenesis in fl wing discs may be blocked due to aberrant expression of the fl gene. Received: 6 November 1996 / Accepted: 5 February 1997     

家蚕miR-2769靶基因的鉴定及表达分析

【目的】MiRNAs在昆虫变态发育过程中发挥非常重要的作用。对家蚕Bombyx mori miRNAs及靶基因的研究将有助于阐明miRNAs参与调控家蚕变态发育的分子机制。【方法】往家蚕5龄第2天幼虫血淋巴注射蜕皮激素20E后,qRT-PCR检测miR-2769在家蚕脂肪体中的表达;通过生物信息学方法预测家蚕miR-2769的靶基因;利用双荧光酶报告载体系统分析miR-2769与预测靶基因BmE75B的互作;qRT-PCR检测miR-2769及其靶基因BmE75不同剪接体在家蚕不同发育时期(幼虫、蛹和成虫)和幼虫不同组织(头、表皮、丝腺、脂肪体、精巢、卵巢、马氏管、中肠和血淋巴)中的表达量。【结果】研究结果表明,miR-2769可通过与家蚕BmE75B的3′UTR区结合位点的互作,显著抑制荧光素酶报告基因的表达。qRT-PCR结果表明,miR-2769和BmE75A/BmE75B在20E诱导家蚕脂肪体中表达趋势相反。时空表达分析结果表明,miR-2769与BmE75的不同剪接体在家蚕不同发育时期和不同组织中均具有特异性表达特征。在家蚕变态发育的不同阶段,miR-2769和BmE75A的表达量均较低,而BmE75B在蛹期表达量较高,BmE75C在5龄末幼虫和蛹早期有极高表达水平。此外,miR-2769与BmE75不同剪接体均存在一定程度的表达负相关。在家蚕5龄幼虫血淋巴中miR-2769可促进BmE75A的表达,在脂肪体中miR-2769可促进BmE75B的表达,而在其他组织中miR-2769抑制BmE75不同剪接体的表达。【结论】家蚕miR-2769可通过与BmE75B的3′UTR区的互作对BmE75不同剪接体的表达进行负调控。     

In vitro effects of juvenile hormone analog on wing disc morphogenesis under ecdysteroid treatment in the female-wingless bagworm moth Eumeta variegata (Insecta: Lepidoptera, Psychidae)

Female adults of the bagworm moth, Eumeta variegata, lack wings completely, whereas male adults of this species have functional wings. We previously found that ecdysteroid induces apoptotic events in the female wing rudiment of E. variegata in vitro, whereas the male wing discs cultured with 20-hydroxyecdysone (20E) underwent apolysis and then cell differentiation. To investigate whether juvenile hormone (JH) in involved in sex-specific cellular response to ecdysteroid during wing development between sexes of E. variegata, we tested the effects of juvenile hormone analog (JHA), methoprene, and 20E on wing disc morphogenesis between sexes in vitro. Using transmission electron microscopy (TEM), we found that both higher concentration of JHA (5 μg/ml) and 20E (1 μg/ml) addition induced cell death (apoptosis) in the male wing discs but not induced cell death in the female wing rudiments in vitro in E. variegata. These culture experiments clearly detected the differential responses of wing discs to JHA under ecdysteroid treatment between sexes. We propose two important hypotheses: (1) JH is not significantly involved in the suppression of the female wing rudiment morphogenesis under 20E treatment, (2) female wing rudiment has lost the ability for cell proliferation in response to the stimulus of 20E.     

DNA甲基化通过调控细胞自噬影响家蚕翅的发育

[目的]本研究旨在探索DNA甲基化是否通过调控细胞自噬进而影响家蚕Bombyx mori翅的发育.[方法]分别用1和2 μg DNA甲基化特异性抑制剂5-aza-dC处理家蚕卵巢Bm12细胞和家蚕预蛹,荧光显微镜下观察Bm12细胞的数量,利用dot blot检测Bm12细胞中DNA甲基化水平,利用溶酶体染色检测细胞自噬...     

The RXR ortholog USP suppresses early metamorphic processes in Drosophila in the absence of ecdysteroids

The steroid hormone 20-hydroxyecdysone (20E) initiates metamorphosis in insects by signaling through the ecdysone receptor complex, a heterodimer of the ecdysone receptor (EcR) and ultraspiracle (USP). Analysis of usp mutant clones in the wing disc of Drosophila shows that in the absence of USP, early hormone responsive genes such as EcR, DHR3 and E75B fail to up-regulate in response to 20E, but other genes that are normally expressed later, such as (&bgr;)-Ftz-F1 and the Z1 isoform of the Broad-Complex (BRC-Z1), are expressed precociously. Sensory neuron formation and axonal outgrowth, two early metamorphic events, also occur prematurely. In vitro experiments with cultured wing discs showed that BRC-Z1 expression and early metamorphic development are rendered steroid-independent in the usp mutant clones. These results are consistent with a model in which these latter processes are induced by a signal arising during the middle of the last larval stage but suppressed by the unliganded EcR/USP complex. Our observations suggest that silencing by the unliganded EcR/USP receptor and the subsequent release of silencing by moderate steroid levels may play an important role in coordinating early phases of steroid driven development.     

Functional analysis of four Gloverin-like genes in the silkworm, Bombyx mori

To identify genes involved in the innate immunity of the silkworm Bombyx mori, we constructed a cDNA library from the fat body of Escherichia coli-challenged B. mori larvae. Based on the expressed sequence tag (EST) data and whole genome shotgun sequence analysis, we found four Gloverin-like genes, BmGlov1-4, in the Bombyx genome. Northern blot and RT-PCR analysis showed that BmGlov1-4 were induced in the larval fat body after an immune challenge by the injection of E. coli; however, less induction was observed after the injection of a yeast Candida albicans. In silico sequence analysis revealed the presence of a motif homologous to NF-kappaB binding site in the upstream region of each BmGlov gene. Moreover, we expressed recombinant BmGlov1-4 proteins using the baculovirus expression system, and found that all the recombinant BmGlov1-4 significantly inhibited the growth of E. coli.