Expression of mdr49 and mdr65 multidrug resistance genes in larval tissues of Drosophila melanogaster under normal and stress conditions

In situ expression of 2 multidrug resistance genes, mdr49 and mdr65, of Drosophila melanogaster was examined in wild-type third instar larval tissues under physiological conditions and after heat shock or colchicine feeding. Expression of these 2 genes was also examined in tumorous tissues of lethal (2) giant larvae I(2)gl4 mutant larvae. These 2 mdr genes show similar constitutive expression in different larval tissues under physiological conditions. However, they are induced differentially by endogenous (tumorous growth) and exogenous stresses (colchcine feeding or heat shock): whereas heat shock and colchicine feeding induce mdr49, tumorous condition is accompanied by enhanced expression of mdr49 and mdr65 genes.     

Insect immunity: developmental and inducible activity of the Drosophila diptericin promoter.

Diptericins are 9 kDa inducible antibacterial peptides initially isolated from immune haemolymph of Phormia (Diptera). Following the isolation of a Drosophila cDNA encoding a diptericin homologue, we have now cloned a genomic fragment containing the Drosophila diptericin gene. To dissect the regulation of this gene, we have transformed flies with a fusion gene in which the reporter beta-galactosidase gene is under the control of 2.2 kb upstream sequences of the diptericin gene. We show that such a fusion gene is inducible by injection of live bacteria or complete Freund's adjuvant and respects the tissue specific expression pattern of the resident diptericin gene. Our analysis reveals at least four distinct phases in the regulation of this gene: young larvae, late third instar larvae, pupae and adults. This complexity may be related to the presence in the upstream sequences of multiple copies of response elements previously characterized in genes encoding acute phase response proteins in mammals (e.g. NK-kappa B, NF-kappa B related, NF-IL6 response elements).     

Targeted gene expression in Drosophila dopaminergic cells using regulatory sequences from tyrosine hydroxylase

Dopamine (DA) is the only catecholaminergic neurotransmitter in the fruit fly Drosophila melanogaster. Dopaminergic neurons have been identified in the larval and adult central nervous system (CNS) in Drosophila and other insects, but no specific genetic tool was available to study their development, function, and degeneration in vivo. In Drosophila as in vertebrates, the rate-limiting step in DA biosynthesis is catalyzed by the enzyme tyrosine hydroxylase (TH). The Drosophila TH gene (DTH) is specifically expressed in all dopaminergic cells and the corresponding mutant, pale (ple), is embryonic lethal. We have performed ple rescue experiments with modified DTH transgenes. Our results indicate that partially redundant regulatory elements located in DTH introns are required for proper expression of this gene in the CNS. Based on this study, we generated a GAL4 driver transgene, TH-GAL4, containing regulatory sequences from the DTH 5' flanking and downstream coding regions. TH-GAL4 specifically expresses in dopaminergic cells in embryos, larval CNS, and adult brain when introduced into the Drosophila genome. As a first application of this driver, we observed that in vivo inhibition of DA release induces a striking hyperexcitability behavior in adult flies. We propose that TH-GAL4 will be useful for studies of the role of DA in behavior and disease models in Drosophila.     

amontillado,the Drosophila homolog of the prohormone processing protease PC2, is required during embryogenesis and early larval development

Biosynthesis of most peptide hormones and neuropeptides requires proteolytic excision of the active peptide from inactive proprotein precursors, an activity carried out by subtilisin-like proprotein convertases (SPCs) in constitutive or regulated secretory pathways. The Drosophila amontillado (amon) gene encodes a homolog of the mammalian PC2 protein, an SPC that functions in the regulated secretory pathway in neuroendocrine tissues. We have identified amon mutants by isolating ethylmethanesulfonate (EMS)-induced lethal and visible mutations that define two complementation groups in the amon interval at 97D1 of the third chromosome. DNA sequencing identified the amon complementation group and the DNA sequence change for each of the nine amon alleles isolated. amon mutants display partial embryonic lethality, are defective in larval growth, and arrest during the first to second instar larval molt. Mutant larvae can be rescued by heat-shock-induced expression of the amon protein. Rescued larvae arrest at the subsequent larval molt, suggesting that amon is also required for the second to third instar larval molt. Our data indicate that the amon proprotein convertase is required during embryogenesis and larval development in Drosophila and support the hypothesis that AMON acts to proteolytically process peptide hormones that regulate hatching, larval growth, and larval ecdysis.     

Sensitivity differences displayed by Drosophila melanogaster larvae of different ages to the toxic effects of growth on media containing aflatoxin B1.

Using Drosophila melanogaster, the relative sensitivities of various larval stages to the toxic effects of growth on media supplemented with either 0.44 or 0.88 ppm aflatoxin B1 (AFB1) were determined. Two strains of fruit flies were tested: strain A-11 which is relatively resistant to AFB1 induced toxicity, and strain A-9 which is quite sensitive. Eggs, mid-first, mid-second and early-, mid- and late-third instar larvae were transferred onto AFB1 media and allowed to complete larval and pupal development and eclose as adults. At the 0.44 ppm concentration, strain A-11 showed no effect, while only first instar larvae of strain A-9 showed significant mortality rates for first instar larvae, but the A-9 larvae die at higher rates than the A-11 larvae. In addition, second and third instar larvae of strain A-9 show significant mortality rates when grown at 0.88 ppm AFB1, while these stages are not affected in strain A-11.     

Tissue-specific developmental requirements of Drosophila tyrosine hydroxylase isoforms

Drosophila tyrosine hydroxylase (DTH) is a key enzyme in dopamine (DA) biosynthesis, which is expressed in neural and hypodermal DA-synthesizing cells. We previously reported that two DTH isoforms are produced in flies through tissue-specific alternative splicing that show distinct regulatory properties. We have now selectively expressed each DTH isoform in vivo in a pale (ple, i.e., DTH-deficient) mutant background. We show that the embryonic lethality of ple can be rescued by expression of the hypodermal, but not the neural, DTH isoform in all DA cells, indicating that the hypoderm- isoform is absolutely required for cuticle biosynthesis and survival in Drosophila. In addition, we report new observations on the consequences of DTH overexpression in the CNS and hypoderm. Our results provide evidence that tissue-specific alternative splicing of the DTH gene is a vital process in Drosophila development.     

Tissue-specific developmental requirements of Drosophila tyrosine hydroxylase isoforms

Drosophila tyrosine hydroxylase (DTH) is a key enzyme in dopamine (DA) biosynthesis, which is expressed in neural and hypodermal DA-synthesizing cells. We previously reported that two DTH isoforms are produced in flies through tissue-specific alternative splicing that show distinct regulatory properties. We have now selectively expressed each DTH isoform in vivo in a pale (ple, i.e., DTH-deficient) mutant background. We show that the embryonic lethality of ple can be rescued by expression of the hypodermal, but not the neural, DTH isoform in all DA cells, indicating that the hypoderm- isoform is absolutely required for cuticle biosynthesis and survival in Drosophila. In addition, we report new observations on the consequences of DTH overexpression in the CNS and hypoderm. Our results provide evidence that tissue-specific alternative splicing of the DTH gene is a vital process in Drosophila development.     

Expression of the heat-shock protein HSP70 in Drosophila buzzatii lines selected for thermal resistance

The level of HSP70 expression induced by a non-lethal high temperature was examined in lines selected for increased thermal resistance and in corresponding control lines of Drosophila buzzatii, in order to test if selection for high temperature resistance leads to an increased level of HSP70 expression. The lines used were selected for up to 64 generations either as adults or through all larval stages. In adult selection lines, hard selection was implemented every second generation after mild heat hardening. In larval selection lines, larvae were exposed each generation to laboratory "natural" selection. Generally lines selected as adults showed a higher HSP70 expression than did controls, both in third instar larvae and in adults. A strong negative response to selection of HSP70 expression was found in all lines that were selected at cycling temperatures during larval development. The results suggests that a trade off between heat resistance in form of HSP70 expression and fecundity/fertility are responsible for the level of HSP70 expression. The effect of the different methods of selection on HSP70 expression suggests that heat resistance constitutes more than one trait.     

Drosophila melanogaster larvae detect low doses of UVC radiation as manifested by a writhing response

Exposure of insects and higher orders of animals to UVC radiation has been shown to result in toxicity with a delayed expression. We have observed an immediate writhing response in slowly wandering third instar larvae of Drosophila melanogaster exposed to UVC radiation at doses that were not lethal. UVB and UVA radiation had no effect. Mutants for the visual and sensory systems appeared to respond normally, but CO₂ anesthesia resulted in reversible inhibition, ebony and silver mutants, which affect different pathways in catecholamine metabolism, showed an absent to reduced response. Using UVC lasers, we were able to demonstrate a response in different regions of the larval body. Furthermore, a 193 nm laser that penetrates only 2–5 μm was able to induce a response but unable to kill the larvae. These results suggest a photochemical reaction occurs in the cuticle which produces free radicals that stimulate the nerves and muscle which are present immediately below the epidermis. Possible targets for the UVC radiation are catecholic compounds secreted and processed into the cuticle of third instar larvae just prior to pupariation whose primary function is to crosslink the protein and carbohydrate components. © 1996 Wiley-Liss, Inc.     

Noncoordinate expression of Drosophila glue genes: Sgs-4 is expressed at many stages and in two different tissues.

The glue genes of Drosophila melanogaster comprise a family of genes expressed at high levels in the salivary glands of late third instar larvae in response to the insect hormone ecdysone. We present evidence that, in contrast to the other glue genes, Sgs-4 is turned on throughout Drosophila development and is not expressed exclusively in the larval salivary glands. Larvae transformed with an Sgs-4/Adh (alcohol dehydrogenase) hybrid gene exhibit Sgs-4-directed Adh expression in the larval proventriculus as well as in the salivary glands as early as the first instar. Sgs-4-specific RNA can be detected at very low levels during all stages of development. During late third instar, levels of Sgs-4 RNA in the salivary glands increase several-thousand-fold, thereby accounting for the large amounts of Sgs-4 protein present in the glue produced by the salivary glands. This pattern of expression is unique to the Sgs-4 gene. While expression of several of the other glue genes can be detected in embryos and early larvae, they appear to be expressed neither throughout development nor in the larval proventriculus. Appearance of the glue gene RNAs in mid third instar salivary glands is noncoordinate, even for the chromosomally clustered genes Sgs-3, Sgs-7, and Sgs-8.     

家蚕酪氨酸羟化酶基因BmTh的表达及功能

酪氨酸羟化酶作为儿茶酚胺合成的限速酶, 广泛存在于昆虫、哺乳动物和人类中, 是其新陈代谢不可缺少的酶类。在其他昆虫中, 酪氨酸羟化酶参与了黑色素的合成, 并在昆虫外骨骼的硬化过程中发挥关键作用。为了研究家蚕Bombyx mori酪氨酸羟化酶基因的生理生化功能, 本文对其基因结构、表达特征及功能进行了研究。基于家蚕基因组和基因芯片数据的生物信息学分析表明, BmTh位于家蚕1号染色体上, 含有8个外显子, 编码561个氨基酸。基因芯片数据显示在家蚕5龄第3天的头部和体壁组织中的表达量较高, RT-PCR验证结果与此一致。利用石蜡组织切片材料和RNA探针对BmTh进行表达定位, 原位杂交结果显示在家蚕头部边缘和体壁上有明显的杂交信号。在幼虫发育至熟蚕时注射酪氨酸羟化酶抑制剂3-indole-L-tyrosine （3-IT）, 20 mmol/L的浓度对幼虫几乎没有影响, 50 mmol/L的浓度导致幼虫变态不完全和化蛹困难, 100 mmol/L的浓度使幼虫致死且体色变黑。结果提示, BmTh对家蚕变态发育起重要作用, 是家蚕正常发育不可缺少的关键基因。     

The dare gene: steroid hormone production, olfactory behavior, and neural degeneration in Drosophila.

Steroid hormones mediate a wide variety of developmental and physiological events in insects, yet little is known about the genetics of insect steroid hormone biosynthesis. Here we describe the Drosophila dare gene, which encodes adrenodoxin reductase (AR). In mammals, AR plays a key role in the synthesis of all steroid hormones. Null mutants of dare undergo developmental arrest during the second larval instar or at the second larval molt, and dare mutants of intermediate severity are delayed in pupariation. These defects are rescued to a high degree by feeding mutant larvae the insect steroid hormone 20-hydroxyecdysone. These data, together with the abundant expression of dare in the two principal steroid biosynthetic tissues, the ring gland and the ovary, argue strongly for a role of dare in steroid hormone production. dare is the first Drosophila gene shown to encode a defined component of the steroid hormone biosynthetic cascade and therefore provides a new tool for the analysis of steroid hormone function. We have explored its role in the adult nervous system and found two striking phenotypes not previously described in mutants affected in steroid hormone signaling. First, we show that mild reductions of dare expression cause abnormal behavioral responses to olfactory stimuli, indicating a requirement for dare in sensory behavior. Then we show that dare mutations of intermediate strength result in rapid, widespread degeneration of the adult nervous system.     

An ultrastructural analysis of the ecdysoneless ((l(3)ecd 1ts) ring gland during the third larval instar of Drosophila melanogaster

Summary In the late third larval instar of Drosophila melanogaster, the prothoracic gland, an endocrine portion of the ring gland, synthesizes ecdysteroids at an accelerated rate. The resultant ecdysteroid titer peak initiates the events associated with metamorphosis. The normal prothoracic gland displays several ultrastructural features at this developmental stage that reflect increased steroidogenic activity, including extensive infoldings of the plasma membrane (membrane invaginations) and an increase in both the concentration of smooth endoplasmic reticulum (SER) (or transitional ER) and elongated mitochondria. By contrast, the prothoracic glands of larvae homozygous for a conditional larval lethal mutation, l(3)ecd ^1ts, not only fail to produce ecdysteroids at normal levels at the restrictive temperature (29° C), but also acquire abnormal morphological features that reflect the disruptive effects of the mutation. These abnormalities include an accumulation of lipid droplets presumed to contain sterol precursors of ecdysteroids, a disappearance of SER and a drastic reduction of membrane invaginations in the peripheral area of the cell. These morphological defects are observed in prothoracic glands dissected from larvae transferred from 18° C to 29° C approximately 24 h before observation and also within 4 h of an in vitro transfer to 29° C following dissection from wandering third instar larvae reared at 18° C. No ultrastructural abnormalities were noted in the corpus allatum portion of mutant ring glands. These observations further indicate the direct involvement of the ecd gene product in ecdysteroid synthesis and suggest a role for the gene in the proper transport of precursors to the site where they can be utilized in ecdysteroid biosynthesis.     

Temporally restricted expression of transcription factor betaFTZ-F1: significance for embryogenesis, molting and metamorphosis in Drosophila melanogaster

FTZ-F1, a member of the nuclear receptor superfamily, has been implicated in the activation of the segmentation gene fushi tarazu during early embryogenesis of Drosophila melanogaster. We found that an isoform of FTZ-F1, betaFTZ-F1, is expressed in the nuclei of almost all tissues slightly before the first and second larval ecdysis and before pupation. Severely affected ftz-f1 mutants display an embryonic lethal phenotype, but can be rescued by ectopic expression of betaFTZ-F1 during the period of endogenous betaFTZ-F1 expression in the wild type. The resulting larvae are not able to molt, but this activity is rescued again by forced expression of betaFTZ-F1, allowing progression to the next larval instar stage. On the other hand, premature expression of betaFTZ-F1 in wild-type larvae at mid-first instar or mid-second instar stages causes defects in the molting process. Sensitive periods were found to be around the time of peak ecdysteroid levels and slightly before the start of endogenous betaFTZ-F1 expression. A hypomorphic ftz-f1 mutant that arrests in the prepupal stage can also be rescued by ectopic, time-specific expression of betaFTZ-F1. Failure of salivary gland histolysis, one of the phenotypes of the ftz-f1 mutant, is rescued by forced expression of the ftz-f1 downstream gene BR-C during the late prepupal period. These results suggest that betaFTZ-F1 regulates genes associated with ecdysis and metamorphosis, and that the exact timing of its action in the ecdysone-induced gene cascade is important for proper development.     

Developmental Arrest and Ecdysteroid Deficiency Resulting from Mutations at the Dre4 Locus of Drosophila

Loss-of-function mutations of the dre4 gene of Drosophila melanogaster caused stage-specific developmental arrest, the stages of arrest coinciding with periods of ecdysteroid (molting hormone) regulated development. Nonconditional mutations resulted in the arrest of larval development in the first instar; embryogenesis was not impaired, and mutant larvae were behaviorally normal and long-lived. At 31 degrees the temperature-sensitive dre4e55 allele caused the arrest of larval development in the first or second instars. When upshifted to 31 degrees at various times during development, dre4e55 mutants exhibited nonpupariation of third-instar larvae, failure of pupal head eversion, failure of adult differentiation, or noneclosion of pharate adults. Under some temperature regimens second-instar larvae pupariated precociously without entering the normally intervening third-instar. Nonpupariation and defects in metamorphosis were associated with the reduction or elimination of ecdysteroid peaks normally associated with late-larval, prepupal, pupal and pharate adult development. Ecdysteroid production by larval ring glands from dre4e55 hemizygous larvae was suppressed after 2 hr of incubation in vitro at 31 degrees, indicating autonomous expression of the dre4 gene in the ring gland. We postulate that the dre4 gene is required for ecdysteroid production at multiple stages of Drosophila development and that the pathologies observed in dre4 mutants reflect developmental consequences of ecdysteroid deficiency.