Ectopic expression of sex-peptide in a variety of tissues in Drosophila females using the P[GAL4] enhancer-trap system

Sex-peptide (SP), which is secreted by the accessory gland of Drosophila males, is transferred to the female during copulation, thereby reducing her sexual appetite (receptivity to males) and stimulating ovulation/oviposition. SP is known to be taken up into the hemolymph of mated females, but it is not clear whether there are two separate target tissues, for behavioral changes and ovulation or only one target for both responses. We have employed the GAL4-UAS system to express SP transgene constructs, both in different tissues and in different cellular components of virgin females. A cytoplasmic form of SP lacking a signal sequence did not evoke any responses, even when expressed ubiquitously. In contrast, a membrane-bound form of SP induced typical post-mating behavior, indicating that SP must be outside the cell in order to exert its biological effects. A total of 204 randomly selected P[GAL4] enhancer-trap lines were screened for their ability to induce SP responses in combination with the membrane-bound SP expressed under GAL4 control. Thirty-three lines were associated with both behavioral change and stimulated ovulation. No line was associated with only one of the two responses, implying that the SP target(s) mediating the two responses are either identical, very closely located, or present in two distinct tissues with a common set of genetic determinants. Western blot analysis of head, thorax, and abdominal extracts revealed that the biological activity was correlated with expression in the head fraction. Received: 3 October 1996 / Accepted: 6 January 1997     

Functional dissection of the neural substrates for sexual behaviors in Drosophila melanogaster

The male-specific Fruitless proteins (FruM) act to establish the potential for male courtship behavior in Drosophila melanogaster and are expressed in small groups of neurons throughout the nervous system. We screened ～1000 GAL4 lines, using assays for general courtship, male-male interactions, and male fertility to determine the phenotypes resulting from the GAL4-driven inhibition of FruM expression in subsets of these neurons. A battery of secondary assays showed that the phenotypic classes of GAL4 lines could be divided into subgroups on the basis of additional neurobiological and behavioral criteria. For example, in some lines, restoration of FruM expression in cholinergic neurons restores fertility or reduces male-male courtship. Persistent chains of males courting each other in some lines results from males courting both sexes indiscriminately, whereas in other lines this phenotype results from apparent habituation deficits. Inhibition of ectopic FruM expression in females, in populations of neurons where FruM is necessary for male fertility, can rescue female infertility. To identify the neurons responsible for some of the observed behavioral alterations, we determined the overlap between the identified GAL4 lines and endogenous FruM expression in lines with fertility defects. The GAL4 lines causing fertility defects generally had widespread overlap with FruM expression in many regions of the nervous system, suggesting likely redundant FruM-expressing neuronal pathways capable of conferring male fertility. From associations between the screened behaviors, we propose a functional model for courtship initiation.     

A Hormone Receptor-Based Transactivator Bridges Different Binary Systems to Precisely Control Spatial-Temporal Gene Expression in Drosophila

The GAL4/UAS gene expression system is a precise means of targeted gene expression employed to study biological phenomena in Drosophila. A modified GAL4/UAS system can be conditionally regulated using a temporal and regional gene expression targeting (TARGET) system that responds to heat shock induction. However heat shock-related temperature shifts sometimes cause unexpected physiological responses that confound behavioral analyses. We describe here the construction of a drug-inducible version of this system that takes advantage of tissue-specific GAL4 driver lines to yield either RU486-activated LexA-progesterone receptor chimeras (LexPR) or β-estradiol-activated LexA-estrogen receptor chimeras (XVE). Upon induction, these chimeras bind to a LexA operator (LexAop) and activate transgene expression. Using GFP expression as a marker for induction in fly brain cells, both approaches are capable of tightly and precisely modulating transgene expression in a temporal and dosage-dependent manner. Additionally, tissue-specific GAL4 drivers resulted in target gene expression that was restricted to those specific tissues. Constitutive expression of the active PKA catalytic subunit using these systems altered the sleep pattern of flies, demonstrating that both systems can regulate transgene expression that precisely mimics regulation that was previously engineered using the GeneSwitch/UAS system. Unlike the limited number of GeneSwitch drivers, this approach allows for the usage of the multitudinous, tissue-specific GAL4 lines for studying temporal gene regulation and tissue-specific gene expression. Together, these new inducible systems provide additional, highly valuable tools available to study gene function in Drosophila.     

Expression level of sarah, a homolog of DSCR1, is critical for ovulation and female courtship behavior in Drosophila melanogaster

To better understand the genetic bases of postmating responses in Drosophila melanogaster females, we screened a collection of P{GS} insertion lines and identified two insertions in sarah (sra), whose misexpression in the nervous system induced high levels of ovulation in virgins. The gene sra encodes a protein similar to human Down syndrome critical region 1 (DSCR1). The ovulation phenotype was reproduced in transgenic virgins expressing UAS-sra in the nervous system. The flies also extruded the ovipositor toward courting males as seen in wild-type mated females, supporting the notion that ovulation and behavioral patterns are physiologically coupled. The sra insertions were found to be hypomorphic alleles with reduced expression levels. Females homozygous for these alleles show: (1) spontaneous ovulation in virgins, (2) sterility with impaired meiotic progression, and (3) compromised postmating responses with lower ovulation level, higher remating rate, and shorter period for restoration of receptivity. No obvious defects were observed in the homozygous males. The gene sra is predominantly expressed in oocytes, nurse cells, and the nervous system. Taken together, these results indicate that the expression level of sra is critical for ovulation and female courtship behavior, including their postmating changes.     

The hydroxyproline motif of male sex peptide elicits the innate immune response in Drosophila females

Seminal fluid elicits a variety of physiological and behavioral changes in insect females. In Drosophila melanogaster females, sex peptide (SP) is the major seminal agent eliciting oviposition and reduction of receptivity. But SP also has many other effects; for example, it stimulates food intake, egg production, ovulation, juvenile hormone production and antimicrobial peptide synthesis. Thus, SP very probably has several receptors. To identify putative targets and signaling cascades, we studied the genome-wide regulation of genes by microarray analysis of RNA isolated from females after mating with wild-type males or males lacking SP, respectively. In addition, we studied the effects of SP on the proteome of females. Sex peptide regulates gene activity differentially in the head and in the abdomen. Genes coding for unspecific antimicrobial peptides are specifically transcribed in the abdomen, e.g. the antimicrobial peptide drosocin in epithelial tissues of the female genital tract (oviduct and calyx). Hence, SP elicits a systemic [Peng J, Zipperlen P & Kubli E (2005) Curr Biol15, 1690-1694] and an epithelial immune response. Ectopic expression of SP in the fat body of transgenic virgin females (with subsequent secretion into the hemolymph) does not elicit drosocin synthesis in the genital tract. Thus, the receptors for the stimulation of the systemic and the epithelial responses by SP are compartmentalized. The hydroxyproline (P*) motif of SP, P*TKFP*IP*SP*NP*, is identified as a novel elicitor of the innate immune response. We suggest that SP acts by chemical mimicry of sugar components of the bacterial cell wall. Thus, SP may induce the immune system via pattern recognition receptors.     

Conversion of lacZ enhancer trap lines to GAL4 lines using targeted transposition in Drosophila melanogaster

Since the development of the enhancer trap technique, many large libraries of nuclear localized lacZ P-element stocks have been generated. These lines can lend themselves to the molecular and biological characterization of new genes. However they are not as useful for the study of development of cellular morphologies. With the advent of the GAL4 expression system, enhancer traps have a far greater potential for utility in biological studies. Yet generation of GAL4 lines by standard random mobilization has been reported to have a low efficiency. To avoid this problem we have employed targeted transposition to generate glial-specific GAL4 lines for the study of glial cellular development. Targeted transposition is the precise exchange of one P element for another. We report the successful and complete replacement of two glial enhancer trap P[lacZ, ry+] elements with the P[GAL4, w+] element. The frequencies of transposition to the target loci were 1.3% and 0.4%. We have thus found it more efficient to generate GAL4 lines from preexisting P-element lines than to obtain tissue-specific expression of GAL4 by random P-element mobilization. It is likely that similar screens can be performed to convert many other P-element lines to the GAL4 system.     

GAL4/UAS系统在转基因技术中的应用研究进展

GAL4/UAS系统是一种转基因技术体系,其原理是利用特定的启动子或增强子,以组织特异性的方式激活酵母转录激活子GAL4的表达,GAL4又以同样的方式引起GAL4反应元件(UAS)-靶基因的转录。GAL4/UAS系统的关键点在于:GAL4基因和UAS-靶基因分别存在于两个转基因系中。GAL4转基因系中有转录激活子,但没有靶基因;在UAS-靶基因系中,转录激活子不存在,因而靶基因处于沉默状态,只有将GAL4转基因系与UAS-靶基因系进行杂交,才可能产生表达靶基因的后代。本文综述了GAL4/UAS系统的建立及其研究应用。     

Galanin in adrenocorticotropic hormone cells is decreased by castration

Galanin (GAL) is a neuropeptide that is widely expressed in neuroendocrine tissues, including the adenohypophysis. Since GAL is expressed at higher levels in females, little attention has been paid to the regulation of GAL secretion in males. Here, we show that testosterone regulates GAL secretion in the adenohypophysis of male rats. Using double immunoelectron microscopy, we demonstrate that GAL cells possess three types of secretory granule: those with colocalization of GAL and adrenocorticotropic hormone (ACTH), those with GAL only, and those with ACTH only. The predominant granule type was that containing both GAL and ACTH, suggesting that GAL and ACTH are secreted at the same time. Indeed, adrenalectomy induced an acute decrease in the number of both GAL cells and ACTH cells. In contrast, castration resulted in a decrease in the proportion of GAL cells, while the proportion of ACTH cells remained unchanged. In addition, ACTH-strongly positive and GAL-positive cells were decreased while ACTH-weakly positive and GAL-negative cells were increased after castration. Testosterone treatment of castrated animals resulted in restoration of these levels to those of intact and sham operated animals. These results indicated that testosterone regulates GAL secretion in male animals.     

Female mating strategy in an enclosed group of Japanese macaques

Female Japanese macaques (Macaca fuscata) are noted for mating with multiple males and for their ability to exert mate choice. In a captive group of Japanese macaques housed at the Primate Research Institute of Kyoto University, Japan, behavioral and endocrine data were combined to examine female mating strategies. During one breeding season, daily behavioral observations were conducted on females who exhibited copulatory behavior. Blood was collected from females twice weekly and their ovulatory periods estimated by analyzing hormone profiles. Females began mating shortly before ovulation, peaked at ovulation, and continued receiving ejaculations for up to ten weeks after conception. Females were more responsible than males for inbreeding avoidance with matrilineal kin. Males sometimes approached females from their own matriline, but females avoided such males and expressed mate choice behavior preferentially toward non-matrilineal males. Over the entire mating season, females did not choose non-matrilineal males on the basis of displays, dominance rank, age, weight, or weight change during the mating season. When females were likely to conceive, however, they expressed mate choice behavior toward males who displayed most frequently. Female mating strategy may include both mate choice at ovulation and other, non-procreative functions.     

Targeted gene expression using the GAL4/UAS system in the silkworm Bombyx mori

The silkworm Bombyx mori is one of the most well-studied insects in terms of both genetics and physiology and is recognized as the model lepidopteran insect. To develop an efficient system for analyzing gene function in the silkworm, we investigated the feasibility of using the GAL4/UAS system in conjunction with piggyBac vector-mediated germ-line transformation for targeted gene expression. To drive the GAL4 gene, we used two endogenous promoters that originated from the B. mori actin A3 (BmA3) and fibroin light-chain (FiL) genes and the artificial promoter 3xP3. GFP was used as the reporter. In initial tests of the function of the GAL4/UAS system, we generated transgenic animals that carried the UAS-GFP construct plus either BmA3-GAL4 or 3xP3-GAL4. GFP fluorescence was observed in the tissues of GFP-positive animals, in which both promoters drove GAL4 gene expression. Animals that possessed only the GAL4 gene or UAS-GFP construct did not show GFP fluorescence. In addition, as a further test of the ability of the GAL4/UAS system to drive tissue-specific expression we constructed FiL-GAL4 lines with 3xP3-CFP as the transformation marker. FiL-GAL4 x UAS-GFP crosses showed GFP expression in the posterior silk gland, in which the endogenous FiL gene is normally expressed. These results show that the GAL4/UAS system is applicable to B. mori and emphasize the potential of this system for controlled analyses of B. mori gene function.     

A green fluorescent protein enhancer trap screen in Drosophila photoreceptor cells

The Drosophila ommatidia contain two classes of photoreceptor cells (PR's), the outer and the inner PR's. We performed an enhancer trap screen in order to target genes specifically expressed in PR's. Using the UAS/GAL4 method with enhanced green fluorescent protein (eGFP) as a vital marker, we screened 180000 flies. Out of 2730 lines exhibiting new eGFP patterns, we focused on 16 lines expressing eGFP in particular subsets of PR's. In particular, we describe three lines inserted near the spalt major, m-spondin and furrowed genes, whose respective expression patterns resemble those genes. These genes had not been reported to be expressed in the adult eye. These examples clearly show the ability of our screen to target genes expressed in the adult Drosophila eye.     

Characterization of Side Populations in HNSCC: Highly Invasive,Chemoresistant and Abnormal Wnt Signaling

Side Population (SP) cells, a subset of Hoechst-low cells, are enriched with stem cells. Originally, SP cells were isolated from bone marrow but recently have been found in various solid tumors and cancer cell lines that are clonogenic in vitro and tumorigenic in vivo. In this study, SP cells from lymph node metastatic head and neck squamous cell carcinoma (HNSCC) cell lines were examined using flow cytometry and Hoechst 3342 efflux assay. We found that highly metastatic HNSCC cell lines M3a2 and M4e contained more SP cells compared to the low metastatic parental HNSCC cell line 686LN. SP cells in HNSCC were highly invasive in vitro and tumorigenic in vivo compared to non-SP cells. Furthermore, SP cells highly expressed ABCG2 and were chemoresistant to Bortezomib and etoposide. Importantly, we found that SP cells in HNSCC had abnormal activation of Wnt/β-catenin signaling as compared to non-SP cells. Together, these findings indicate that SP cells might be a major driving force of head and neck tumor formation and metastasis. The Wnt/β-catenin signaling pathway may be an important target for eliminating cancer stem cells in HNSCC.     

Larval chemosensory projections and invasion of adult afferents in the antennal lobe of Drosophila

We have studied the fate of olfactory afferents during metamorphic transformation of Drosophila melanogaster. Intracellular labeling of afferents from larval head chemosensilla suggests that the larval antennal lobe may be an olfactory target, whereas tritocerebral and suboesophageal centers are likely targets of gustatory sensilla. Application of monoclonal antibody 22C10 shows that the larval antennal nerve is the precursor of the adult antennal nerve and is used as a centripetal pathway for the adult afferents. Likely guidance cues are larval olfactory afferents that persist during early metamorphosis. P[GAL4] enhancer trap lines are introduced as efficient markers to follow the establishment of adult sensory projections. β-Galactosidase and the bovine TAU protein were used as reporter proteins, and their expression patterns are compared. P[GAL4] lines MT14 and KL116 demonstrate that adult antennal afferents have arrived in the antennal lobe 24 h after pupariation and extend to the contralateral lobe 6 h later. Line MT14 expresses GAL4 mostly in basiconic sensilla and in certain trichoid sensilla, whereas KL116 is specific for trichoid and a small subset of basiconic sensilla. In the antennal lobe, largely complementary subsets of glomeruli are labeled by the two lines, in agreement with the observation that particular types of sensilla project to particular target glomeruli. © 1997 John Wiley & Sons, Inc. J Neurobiol 32: 281–297, 1997.     

Knockdown of LINC01116 inhibits cell migration and invasion in head and neck squamous cell carcinoma through epithelial-mesenchymal transition pathway

Long noncoding RNAs (lncRNAs) are linked to tumor development and progression. The aim of this study was to determine the prognostic significance and biological role of LINC01116 in head and neck squamous cell carcinoma (HNSCC). We identified 21 aberrantly expressed lncRNAs specific to HNSCC that were common in two microarray datasets. LINC01116 was highly overexpressed in HNSCC tissues and was correlated to shorter overall survival and relapse-free survival duration, as analyzed by the online Gene Expression Profiling Interactive Analysis platform. LINC01116 was also overexpressed in oral squamous cell carcinoma and nasopharyngeal carcinoma tissues, and LINC01116 silencing significantly inhibited the migration and invasion capacities of both cell lines by blocking the epithelial-mesenchymal transition process. In addition, 125 coexpressing genes were identified by circlncRNAnet, and were mainly located on human autosomes and enriched in transforming growth factor-β signaling pathway. These findings indicate that LINC01116 might be a potential therapeutic target for HNSCC.     

Combination of the ALCR/alcA ethanol switch and GAL4/VP16-UAS enhancer trap system enables spatial and temporal control of transgene expression in Arabidopsis

The experimental control of gene expression in specific tissues or cells at defined time points is a useful tool for the analysis of gene function. GAL4/VP16-UAS enhancer trap lines can be used to selectively express genes in specific tissues or cells, and an ethanol-inducible system can help to control the time of expression. In this study, the combination of the two methods allowed the successful regulation of gene expression in both time and space. For this purpose, a binary vector, 962-UAS::GUS, was constructed in which the ALCR activator and β-glucuronidase (GUS) reporter gene were placed under the control of upstream activator sequence (UAS) elements and the alcA response element, respectively. Three different GAL4/VP16-UAS enhancer trap lines of Arabidopsis were transformed, resulting in transgenic plants in which GUS activity was detected only on ethanol induction and exclusively in the predicted tissues of the enhancer trap lines. As a library of different enhancer trap lines with distinct green fluorescent protein (GFP) patterns exist, transformation with a similar vector, in which GUS is replaced by another gene, would enable the control of the time and place of transgene expression. We have constructed two vectors for easy cloning of the gene of interest, one with a polylinker site and one that is compatible with the GATEWAY™ vector conversion system. The method can be extended to other species when enhancer trap lines become available.     

Fixation and locomotor activity are impaired by inducing tetanus toxin expression in adult Drosophila brain

Visual fixation and locomotor activity are two important behavioral properties utilized by flies when they approach a landmark. Although previous studies in Drosophila have revealed that the mushroom bodies (Mbs) and the central complex (CC) were regulatory centers for these behaviors, the specific neurons involved still remain largely unknown. We tested visual fixation behavior and locomotor activity of flies in a simple choice assay, Buridan's paradigm, using the GAL4/UAS system to express tetanus toxin light chain (TeTxLC) in adult neurons specifically. Although we explored a variety of mushroom body and central complex-labeling lines, we found that only four GAL4 lines (104y-GAL4, 121y-GAL4, 154y-GAL4 and 210y-GAL4) could produce significant defects in fixation as well as decrease locomotor activity following adult induction of TeTxLC. This suggests a more complex circuit is involved in controlling these behaviors than previously thought. Expression patterns of the GAL4 lines in the central nervous system provide the some clues to which neurons might be involved in this neural circuit.