Activated Ras/JNK driven Dilp8 in imaginal discs adversely affects organismal homeostasis during early pupal stage in Drosophila,a new checkpoint for development

Background

Dilp8-mediated inhibition of ecdysone synthesis and pupation in holometabolous insects maintains developmental homeostasis through stringent control of timing and strength of molting signals. We examined reasons for normal pupation but early pupal death observed in certain cases.

Results

Overexpression of activated Ras in developing eye/wing discs inhibited Ptth expression in brain via upregulated JNK signaling mediated Dilp8 secretion from imaginal discs, which inhibited ecdysone synthesis in prothoracic gland after pupariation, leading to death of ~25- to 30-hour-old pupae. Inhibition of elevated Ras signaling completely rescued early pupal death while post-pupation administration of ecdysone to organisms with elevated Ras signaling in eye discs partially rescued their early pupal death. Unlike the earlier known Dilp8 action in delaying pupation, hyperactivated Ras mediated elevation of pJNK signaling in imaginal discs caused Dilp8 secretion after pupariation. Ectopic expression of certain other transgene causing pupal lethality similarly enhanced pJNK and early pupal Dilp8 levels. Suboptimal ecdysone levels after 8 hours of pupation prevented the early pupal metamorphic changes and caused organismal death.

Conclusions

Our results reveal early pupal stage as a novel Dilp8 mediated post-pupariation checkpoint and provide further evidence for interorgan signaling during development, wherein a peripheral tissue influences the CNS driven endocrine function.

     

Localization of calcium ions in mixed synapses of Mauthner neurons during exposure to substances altering the conductivity of gap junctions

The pyroantimonate method was used to study the distribution of calcium ions in the mixed synapses of Mauthner neurons after exposure to substances altering the electrotonic conductivity of these synapses mediated by gap junctions (GJ). Ecdysone, an agent which increases GJ conductivity, produced precipitates of calcium pyroantimonate coating the whole postsynaptic surface of the GJ area, making them strongly asymmetrical. Precipitate granules were also seen to appear in the clefts of desmosome-like contacts (DLC). Chlorpromazine, which decreases GJ conductivity, produced precipitates in GJ clefts and on the pre- and postsynaptic membranes. No precipitate formed in DLC clefts. These results demonstrate that ecdysone acts as an agent selectively increasing GJ conductivity without affecting DLC function. Chlorpromazine had a double action, blocking conduction through both GJ and DLC. Thus, studies of agents altering GJ permeability require consideration of the possibility that they may interact with actin-containing structures also involved in the transport of the electrotonic signal.Translated from Morfologiya, Vol. 125, No. 3, pp. 32–35, May–June, 2004.     

The P450 enzyme Shade mediates the hydroxylation of ecdysone to 20‐hydroxyecdysone in the Colorado potato beetle,Leptinotarsa decemlineata

Ecdysone 20‐monooxygenase (E20MO), a cytochrome P450 monooxygenase (CYP314A1), catalyses the conversion of ecdysone (E) to 20‐hydroxyecdysone (20E). We report here the cloning and characterization of the Halloween gene Shade (Shd) encoding E20MO in the Colorado potato beetle, Leptinotarsa decemlineata. LdSHD has five conserved motifs typical of insect P450s, ie the Helix‐C, Helix‐I, Helix‐K, PxxFxPE/DRF (PERF) and heme‐binding motifs. LdShd was expressed in developing eggs, the first to fourth instars, wandering larvae, pupae and adults, with statistically significant fluctuations. Its mRNA was ubiquitously distributed in the head, thorax and abdomen. The recombinant LdSHD protein expressed in Spodoptera frugiperda 9 (Sf9) cells catalysed the conversion of E to 20E. Dietary introduction of double‐stranded RNA (dsRNA) of LdShd into the second instar larvae successfully knocked down the LdShd expression level, decreased the mRNA level of the ecdysone receptor (LdEcR) gene, caused larval lethality, delayed development and affected pupation. Moreover, ingestion of LdShd‐dsRNA by the fourth instars also down‐regulated LdShd and LdEcR expression, reduced the 20E titre, and negatively influenced pupation. Introduction of 20E and a nonsteroidal ecdysteroid agonist halofenozide into the LdShd‐dsRNA‐ingested second instars, and of halofenozide into the LdShd‐dsRNA‐ingested fourth instars almost completely relieved the negative effects on larval performance. Thus, LdSHD functions to regulate metamorphotic processes by converting E to 20E in a coleopteran insect species Le. decemlineata.     

受控型蜕皮激素诱导表达截短型hIGF-Ⅰ转基因载体的构建及其在细胞水平上的有效性检测

目的：构建一种可以在哺乳动物细胞中蜕皮激素诱导表达截短型hIGF-Ⅰ的受控型转基因载体，为制备受控型蜕皮激素诱导表达截短型MGF-Ⅰ的转基因小鼠奠定基础。方法：利用分子克隆技术构建受控型蜕皮激素诱导表达截短型hIGF-Ⅰ的转基因载体；将其电转至AM1菌中，利用其中的Cre重组酶将载体上两个同向LoxP序更锚定的新霉素（neomycin）基因删除，解除其对蜕皮激素诱导表达系统的阻断作用，利用PCR、酶切和测序鉴定删除情况；将重组后的载体转染至COS7细胞中进行瞬间表达，蜕皮激素诱导后回收培养上清和细胞裂解物进行Western印迹分析，检测hIGF-Ⅰ的表达情况。结果和结论：成功构建大小为13．6kb的转基因载体pOE-IGF-Ⅰ；转化至AM1中后，PCR、酶切和测序的结果都证明其中的Cre酶能够将载体上neomycin基因删除；重组后的载体转染至COS7细胞中进行诱导表达，Western印迹实验证明截短型MGF-Ⅰ能够在COS7细胞中顺利表达。上述结果证明该蜕皮激素诱导表达截短型hIGF-Ⅰ的受控型转基因载体能够用于转基因小鼠的制备。     

蜕皮激素对顺铂诱导小鼠肝损伤的保护作用及机制研究

目的:探讨蜕皮激素对顺铂导致小鼠肝损伤的保护作用及机制。方法:采用昆明小鼠和AML12细胞构建顺铂肝损伤体内及体外模型,应用蜕皮激素对顺铂组进行干预,通过HE染色观察肝脏组织病理形态,ELISA和荧光染色观测细胞内ROS的产生及细胞凋亡,RT-qPCR检测组织及细胞内氧化应激、炎症和凋亡相关生物标志物的表达。转录组测序测定其作用靶点,RT-qPCR和Western blot验证靶基因mRNA和蛋白表达。结果:与空白组相比,顺铂组小鼠肝细胞排列疏松,部分细胞轻度肿胀,凋亡细胞数量、血清内ALT、AST、肝细胞内ROS、MDA含量明显增多（P<0.01）,GSH-PX、SOD含量明显降低（P<0.01）,提示顺铂对肝功能造成明显损伤;与顺铂组相比,蜕皮激素组小鼠肝细胞损伤程度明显减轻,上述各项指标均有不同程度改善,表明其对肝功能具有保护作用。RT-qPCR结果显示,蜕皮激素组NF-κB、TNF-α、IL-1β、Caspase-3、Caspase-9、Hmox1、PI3K、Nqo1 mRNA相对表达量均明显降低,Nrf2 mRNA相对表达量明显升高（P<0.01）。转录组测...     

Conversion of a radiolabelled ecdysone precursor, 2,22,25-trideoxyecdysone, by embryonic and larval tissues of Locusta migratoria

A high specific activity tritiated ecdysone precursor, 2,22,25-trideoxyecdysone, was used to probe the capacity of various embryonic and larval tissues to perform the last 3 hydroxylation steps in ecdysone biosynthesis. Embryos at early stages of development, prior to the differentiation of their endocrine glands and embryonic heads, thoraces and abdomens of later stages, were found to have the capacity to hydroxylate the precursor to ecdysone. Larval epidermis and fat body are also able to transform 2,22,25-trideoxyecdysone into ecdysone; Malpighian tubules and midgut hydroxylate the precursor at C-2 but are apparently unable to hydroxylate both at C-22 and C-25. Larval prothoracic glands convert the precursor to ecdysone at a very efficient rate, which is 1-2 magnitudes higher than that of the other tissues investigated; several data argue for the existence of a privileged sequence of hydroxylations, C-25, C-22, C-2, in the larval prothoracic glands.     

Ras signaling modulates activity of the ecdysone receptor EcR during cell migration in the Drosophila ovary.

Ecdysone Receptor (EcR) mediates effects of the hormone ecdysone during larval molts, pupal metamorphosis, and adult female oogenesis. In the ovary, egg chamber formation requires interactions between the somatic follicle cell (FC) epithelium and the germ line nurse cell/oocyte cyst. Previous work has shown EcR is required in the germ line for egg chamber maturation, and here we examine EcR requirements in the FC at late stages of oogenesis. EcR protein is ubiquitous in the FC but its activity is restricted, visualized by activity of the "ligand sensor" hs-GAL4-EcR ligand binding domain fusion and EcRE-lacZ reporter gene expression. GAL4-EcR is activated in the FC by an ecdysone agonist and repressed by tissue-specific Ras GTPase signals. To determine the significance of restricted sites of EcR activity in the FC, we used targeted misexpression of the dominant negative EcR (EcR-DN) molecules EcR(F645A) and EcR(W650A). EcR-DN expression at stage 10 reduced EcRE-lacZ expression in the nurse cell FC and resulted in abnormal FC migrations, including aberrant centripetal migration and dorsal appendage tube formation, leading to the formation of cup-shaped eggs with shortened, branched dorsal appendages at stage 14. Clones of FC expressing EcR-DN displayed cell-autonomous increases in DE-cadherin expression and abnormal epithelial junction formation. EcR-DN expression caused thin eggshell phenotypes that correlated with both reduced levels of chorion gene expression and reduction in chorion gene amplification. Our results indicate that tissue-specific modulation of EcR activity by the Ras signaling pathway refines temporal ecdysone signals that regulate FC differentiation and cadherin-mediated epithelial cell shape changes.     

The circadian clock gates Drosophila adult emergence by controlling the timecourse of metamorphosis

The daily rhythm of adult emergence of holometabolous insects is one of the first circadian rhythms to be studied. In these insects, the circadian clock imposes a daily pattern of emergence by allowing or stimulating eclosion during certain windows of time and inhibiting emergence during others, a process that has been described as “gating.” Although the circadian rhythm of insect emergence provided many of the key concepts of chronobiology, little progress has been made in understanding the bases of the gating process itself, although the term “gating” suggests that it is separate from the developmental process of metamorphosis. Here, we follow the progression through the final stages of Drosophila adult development with single-animal resolution and show that the circadian clock imposes a daily rhythmicity to the pattern of emergence by controlling when the insect initiates the final steps of metamorphosis itself. Circadian rhythmicity of emergence depends on the coupling between the central clock located in the brain and a peripheral clock located in the prothoracic gland (PG), an endocrine gland whose only known function is the production of the molting hormone, ecdysone. Here, we show that the clock exerts its action by regulating not the levels of ecdysone but that of its actions mediated by the ecdysone receptor. Our findings may also provide insights for understanding the mechanisms by which the daily rhythms of glucocorticoids are produced in mammals, which result from the coupling between the central clock in the suprachiasmatic nucleus and a peripheral clock located in the suprarenal gland.

Circadian clocks impose a daily rhythmicity to the behavior and physiology of most multicellular organisms, in which they are believed to provide a mechanism for synchronizing the behavior and physiology of individuals to the daily planetary changes in light and temperature (1). One of the first circadian rhythms to be studied is the rhythm of adult emergence of holometabolous insects (eclosion) (2–4). Eclosion occurs at the end of metamorphosis and is typically restricted to dawn or dusk, depending on the species. Population eclosion profiles show a daily rhythmicity that persists under conditions of constant darkness and temperature and is temperature-compensated, thereby showing the hallmarks of a circadian rhythm (e.g., refs. 4 to 6).In holometabolous insect species in which this rhythm has been studied, the circadian clock controls the timing of adult emergence by exerting its influence at the very end of adult development. For example, Pittendrigh and Skopik (6) demonstrated in Drosophila victoria that overt developmental markers for the progression through metamorphosis, such as the time of eye or bristle pigmentation, occur at times that only depend on the number of hours since the start of metamorphosis and are not affected by changes in the light:dark (LD) cycle. By contrast, eclosion (the final step of metamorphosis) is the only event whose timing is sensitive to the LD regime, with flies always emerging during the morning. Similarly, in Drosophila melanogaster, Qiu and Hardin (7) followed the timing of wing pigmentation to show that animals that pigment their wings during the day will primarily emerge during the following day, whereas those that do so at night are delayed an extra ca. 12 h and wait until the light period 2 d later, indicating that the clock exerts its control after the wings have pigmented, during the final day of metamorphosis. Thus, in order to eclose, the insect must have completed metamorphosis and also be within the appropriate time window.Because the clock intervenes at the end of adult development, the process through which it controls the time of emergence has been described as “gating,” in which emergence is inhibited during certain windows of time and stimulated and/or allowed during others. Although the circadian rhythm of eclosion was one of the first to be studied starting almost 100 y ago (2–4), there is still no clear mechanistic understanding of how this gating process occurs. Nevertheless, so far it has been viewed as a process that is independent of any developmental process. In this scenario, animals that completed metamorphosis prematurely would be prevented from eclosing until the appropriate eclosion gate were opened by the circadian clock (5, 6, 8). However, another possibility is that the clock sets the time of emergence by controlling the timecourse of completion of metamorphosis. Although both scenarios would produce a gated eclosion, they differ mechanistically in fundamental ways. In particular, the first “permissive” gating mechanism predicts that animals could complete metamorphosis at different times prior to emergence and that the clock would then only open or close the window leading to eclosion, depending on the time of the day. By contrast, a “developmental” gating mechanism predicts that animals emerging within a specific gate would speed up or slow down their molt in order to complete metamorphosis in time to emerge during the appropriate gate. In this scenario, gating would be a consequence of the synchronization of metamorphosis. Since in at least some insect species, the titers of the molting hormone, ecdysone (E), express a circadian rhythm (9, 10), a clock control of metamorphosis is a plausible scenario.Here, we analyzed the timecourse of the final stages of Drosophila metamorphosis with single-animal resolution and show that the clock gates eclosion by regulating, primarily, the moment when the final steps of metamorphosis are initiated. We also show that the clock exerts its action by regulating not the levels of the molting hormone itself but that of its actions mediated by the E receptor. Our findings may also provide insights for understanding the mechanisms by which the daily rhythms of glucocorticoid (GC) are produced in mammal, which also depend on the coupling between a central clock, located in the suprachiasmatic nucleus (SCN), and a peripheral clock, located in the suprarenal endocrine gland (11).