Larval Salivary Glands Research Articles

Proteomic Identification of PKC-Mediated Expression of 20E-Induced Protein in Drosophila melanogaster

Ecdysone receptor (EcR) and its heterodimeric partner, ultraspiracle protein (USP), are nuclear receptors that mediate the action of the insect molting hormone 20-hydroxyecdysone (20E). There is evidence that the activity of both receptors is affected by phosphorylation. Using a proteomic approach, we have shown that protein kinase C (PKC) activity is necessary for mediating 20E-induced expression of 14 specific proteins, including three previously reported 20E responsive proteins, and is also responsible for the intracellular localization of EcR and USP in larval salivary glands of Drosophila melanogaster. The 20E-dependent expression of the proteins was verified using real-time PCR and/or Western blot analysis. For some genes, inhibition of PKC activity reduced 20E-dependent transcriptional activity rapidly, raising the possibility that these are direct gene targets of EcR and USP. The data further indicate that PKC-mediated phosphorylation is also required for genes regulated indirectly by 20E-induced changes in the larval salivary gland.

Tissue-dependent subcellular localization of Drosophila arginine methyl-transferase 4 (DART4), a coactivator whose overexpression affects neither viability nor differentiation

Drosophila arginine methyl- transferase 4 (DART4) belongs to the type I class of arginine methyltransferases. It catalyzes the methylation of arginine residues to monomethylarginines and asymmetrical dimethylarginines. The DART4 sequence is highly similar to mammalian PRMT4/CARM1, and DART4 substrate specificity has been conserved, too. Recently it was suggested that DART4/Carmer functions in ecdysone receptor mediated apoptosis of the polytene larval salivary glands and an apparent up-regulation of DART4/ Carmer mRNA levels before tissue histolysis was reported. Here we show that in Drosophila larvae, DART4 is mainly expressed in the imaginal disks and in larval brains, and to a much lesser degree in the polytene larval tissue such as salivary glands. In glands, DART4 protein is present in the cytoplasm and the nucleus. The nuclear signal emanates from the extrachromosomal domain and gets progressively restricted to the region of the nuclear lamina upon pupariation. Surprisingly, DART4 levels do not increase in salivary glands during pupariation, and overexpression of DART4 does not cause precautious cell death in the glands. Furthermore, over- and misexpression of DART4 under the control of the α tubulin promoter do not lead to any major problem in the life of a fly. This suggests that DART4 activity is regulated at the posttranslational level and/or that it acts as a true cofactor in vivo. We present evidence that nuclear localization of DART4 may contribute to its function because DART4 accumulation changes from a distribution with a strong cytoplasmic component during the transcriptional quiescence of the young embryo to a predominantly nuclear one at the onset of zygotic transcription.

Caspase‐dependent apoptosis induction by targeted expression of DEK in drosophila involves histone acetylation inhibition

As a nuclear phosphoprotein, proto-oncogene protein DEK is capable to changing chromatin structure. DEK was recently identified as an inhibitor of histone acetylation mediated by p300 and PCAF and to facilitate transcriptional repression. To elucidate the biological functions of DEK in vivo, we have constructed transgenic flies that overexpress the human DEK in the developing eye. Transgenic flies developed a severe rough eye phenotype, which is indicative of ectopically induced apoptosis. Genetic and biochemical analyses, including the rescue of the apoptotic phenotype by pan-caspase inhibitor protein p35 and caspase activity analyses, suggested that DEK induces apoptotic cell death through a caspases-9 and -3 dependent pathway. Using extracts from larval salivary glands, we have determined that the global histone acetylation levels of histone H3 Lys9 and H4 Lys5 were decreased upon DEK overexpression. Using chromatin immunoprecipitation assays, we have demonstrated that overexpression of DEK induced the histone H3 and H4 hypoacetylation of promoter of the antiapoptotic gene bcl-2. Co-expression of bcl-2 also rescued apoptosis and the reduced expression of bcl-2 gene was analyzed by real-time PCR. Our results indicate that acidic domain containing protein DEK might have a role in modulating both transcriptional regulation and apoptosis through HAT inhibitory activity.

Normal regulation of Rbf1/E2f1 target genes in Drosophila type 1 protein phosphatase mutants

G1 Cyclin/Cdk complexes phosphorylate and inactivate the pRb tumor suppressor by preventing its ability to bind and repress E2F transcription factors. Current molecular and biochemical evidence suggests that type 1 protein phosphatases (PP1) dephosphorylate and thereby activate pRb, but the functional significance of this has not been addressed in the context of animal development. Here, we use genetic analyses to determine the role of PP1 in the regulation of Rbf1 activity during Drosophila development. While Rbf1 is required for E2f1 inhibition and G1 arrest in the embryonic epidermis and for the periodic expression of E2f1 target genes during endocycle S phase in the embryonic midgut and larval salivary gland, PP1 is not. PP1 regulates periodic cyclin E protein accumulation in ovarian nurse cells independently of Rbf1, which is dispensable for endocycle regulation in this tissue. We conclude that PP1 is not a major regulator of the Rbf1/E2F1 pathway in Drosophila.

Down-regulation of inhibitor of apoptosis levels provides competence for steroid-triggered cell death

A pulse of the steroid hormone ecdysone triggers the destruction of larval salivary glands during Drosophila metamorphosis through a transcriptional cascade that converges on reaper (rpr) and head involution defective (hid) induction, resulting in caspase activation and cell death. We identify the CREB binding protein (CBP) transcriptional cofactor as essential for salivary gland cell death. We show that CBP acts 1 d before the onset of metamorphosis in apparent response to a mid-third instar ecdysone pulse, when CBP is necessary and sufficient for down-regulation of the Drosophila inhibitor of apoptosis 1 (DIAP1). It is only after DIAP1 levels are reduced that salivary glands become competent to die through rpr/hid-mediated cell death. Before this time, high levels of DIAP1 block salivary gland cell death, even in the presence of ectopic rpr expression. This study shows that naturally occurring changes in inhibitor of apoptosis levels can be critical for regulating cell death during development. It also provides a molecular mechanism for the acquisition of competence in steroid signaling pathways.

The extrachromosomal East protein of Drosophila can associate with polytene chromosomes and regulate gene expression.

The EAST protein of Drosophila is a component of an expandable extrachromosomal domain of the nucleus. To better understand its function, we studied the dynamics and localization of GFP-tagged EAST. In live larval salivary glands, EAST-GFP is highly mobile and localizes to the extrachromosomal nucleoplasm. When these cells are permeabilized, EAST-GFP rapidly associated with polytene chromosomes. The affinity to chromatin increases and mobility decreases with decreasing salt concentration. Deleting the C-terminal residues 1535 to 2301 of EAST strongly reduces the affinity to polytene chromosomes. The bulk of EAST-GFP co-localizes with heterochromatin and is absent from transcriptionally active chromosomal regions. The predominantly chromosomal localization of EAST-GFP can be detected in non-detergent treated salivary glands of pupae as they undergo apoptosis, however not in earlier stages of development. Consistent with this chromosomal pattern of localization, genetic evidence indicates a role for EAST in the repression of gene expression, since a lethal east mutation is allelic to the viable mutation suppressor of white-spotted. We propose that EAST acts as an ion sensor that modulates gene expression in response to changing intracellular ion concentrations.

POSTEMBRYONIC DEVELOPMENT OF DROSOPHILA MELANOGASTER MEIGEN, 1830 UNDER THE INFLUENCE OF QUERCETIN

Quercetin is one of the best studied and most abundant flavonoid molecules in plants. Flavonoids are a large group of natural polyphenols that are almost ubiquitously present in the plants. The consumption of fruit and vegetables containing quercetin has been associated with several health benefits. In this study, we examined the impact of dietary quercetin (0, 0.005, 0.05, 0.5 and 1.75% quercetin by weight) on Drosophila melanogaster Meigen, 1830 postembryonic development and histological changes in larvae and females. A possible hormonmimetic (ecdysonergic) activity of quecetin was also studied on polytene chromosomes present in the larval salivary glands. We noticed a decrease in the duration of the larval period of flies raised on 1.75% quercetin diet. Quercetin-fed larvae entered metamorphosis sooner than the control ones. The most dramatic change was observed in larval fat body. Dark round structures were observed in fat body cells from larvae treated with quercetin.

Fork head controls the timing and tissue selectivity of steroid-induced developmental cell death

Cell death during Drosophila melanogaster metamorphosis is controlled by the steroid hormone 20-hydroxyecdysone (20E). Elements of the signaling pathway that triggers death are known, but it is not known why some tissues, and not others, die in response to a particular hormone pulse. We found that loss of the tissue-specific transcription factor Fork head (Fkh) is both required and sufficient to specify a death response to 20E in the larval salivary glands. Loss of fkh itself is a steroid-controlled event that is mediated by the 20E-induced BR-C gene, and that renders the key death regulators hid and reaper hormone responsive. These results implicate the D. melanogaster FOXA orthologue Fkh with a novel function as a competence factor for steroid-controlled cell death. They explain how a specific tissue is singled out for death, and why this tissue survives earlier hormone pulses. More generally, they suggest that cell identity factors like Fkh play a pivotal role in the normal control of developmental cell death.

To die or not to die—a role for Fork head

The precise determination of when and where cells undergo programmed cell death is critical for normal development and tissue homeostasis. Cao et al. (2007; see p. 843 of this issue) report that the Fork head (Fkh) transcription factor, which is essential for the early development and function of the larval salivary glands in Drosophila melanogaster, also contributes to its demise. These authors show that fkh expression in the salivary glands is normally lost at puparium formation, which is ∼12 h before they undergo massive cell death triggered by the steroid hormone ecdysone, making room for their developing adult counterparts. The loss of Fkh eliminates its role in blocking cell death, allowing for subsequent ecdysone-induced reaper and head involution defective death activator expression and tissue destruction. This study provides new insights into the transcriptional regulation of programmed cell death and the mechanisms that underlie the precise spatial and temporal control of hormone responses during development.

Programmed cell death in the larval salivary glands of Apis mellifera (Hymenoptera, Apidae)

The morphological and histochemical features of degeneration in honeybee (Apis mellifera) salivary glands were investigated in 5th instar larvae and in the pre-pupal period. The distribution and activity patterns of acid phosphatase enzyme were also analysed. As a routine,the larval salivary glands were fixed and processed for light microscopy and transmission electron microscopy.Tissue sections were subsequently stained with haematoxylin -eosin,bromophenol blue,silver,or a variant of the critical electrolyte concentration (CEC) method.Ultrathin sections were contrasted with uranyl acetate and lead citrate.Glands were processed for the histochemical and cytochemical localization of acid phosphatase,as well as biochemical assay to detect its activity pattern. Acid phosphatase activity was histochemically detected in all the salivary glands analysed.The cytochemical results showed acid phosphatase in vesicles, Golgi apparatus and lysosomes during the secretory phase and,additionally, in autophagic structures and luminal secretion during the degenerative phase. These findings were in agreement with the biochemical assay. At the end of the 5th instar, the glandular cells had a vacuolated cytoplasm and pyknotic nuclei, and epithelial cells were shed into the glandular lumen.The transition phase from the 5th instar to the pre-pupal period was characterized by intense vacuolation of the basal cytoplasm and release of parts of the cytoplasm into the lumen by apical blebbing; these blebs contained cytoplasmic RNA, rough endoplasmic reticule and, occasionally, nuclear material. In the pre-pupal phase, the glandular epithelium showed progressive degeneration so that at the end of this phase only nuclei and remnants of the cytoplasm were observed.The nuclei were pyknotic,with peripheral chromatin and blebs. The gland remained in the haemolymph and was recycled during metamorphosis. The programmed cell death in this gland represented a morphological form intermediate between apoptosis and autophagy.

Proteomic analysis of steroid-triggered autophagic programmed cell death during Drosophila development

Two morphological forms of programmed cell death, apoptosis and autophagic cell death, remove unneeded or damaged cells during animal development. Although the mechanisms that regulate apoptosis are well studied, little is known about autophagic cell death. A shotgun proteome analysis of purified dying larval salivary glands in Drosophila was used to identify proteins that are expressed during autophagic programmed cell death. A total of 5661 proteins were identified from stages before and after the onset of cell death. Analyses of these data enabled us to identify proteins from a number of interesting categories including regulators of transcription, the apoptosis, autophagy, lysosomal, and ubiquitin proteasome degradation pathways, and proteins involved in growth control. Several of the identified proteins, including the serine/threonine kinase warts (Wts), were not detected using whole-genome DNA microarrays, providing support for the importance of such high-throughput proteomic technology. Wts regulates cell-cycle arrest and apoptosis, and significantly, mutations in wts prevent destruction of salivary glands.

Functional genomics of histone modification and non-histone chromosomal proteins using the polytene chromosomes of Drosophila

Knowing the genomic distribution of chromosomal proteins and of histone modifications provides essential insight into function. The giant polytene chromosomes of the Drosophila larval salivary glands provide a high-resolution genomic map with a resolution exceeded only by chromatin immunoprecipitation. Immunofluorescence localization of chromosomal proteins and specific post-translational modifications of histones is a simple and rapid tool for the functional genomics of chromosomal proteins.

Genome size and determination of DNA content of the X chromosomes, autosomes, and germ line‐limited chromosomes of Sciara coprophila

The unique chromosome biology of the fungus fly Sciara coprophila has fascinated investigators for over 80 years. Male meiosis exhibits a monopolar spindle, nonrandom segregation of imprinted chromosomes and nondisjunction of the X chromosome. The unusual mechanism of sex determination requires selective elimination of X chromosomes in embryogenesis. Supernumerary (L) chromosomes are also eliminated from the soma during early cleavage divisions. Distinctive DNA puffs on the larval salivary gland chromosomes are sites of DNA amplification. As a foundation for future genome studies to explore these many unusual phenomena, we have used DNA-Feulgen cytophotometry to determine genome size from hemocyte nuclei of male (X0) and female (XX) larvae and adults. The DNA content of the X chromosome is approximately 0.05 pg DNA and the autosomal complement is approximately 0.45 pg DNA. Measurements of DNA levels for individual sperm from adults showed that the DNA contribution of the germ line-limited (L) chromosomes constitutes as much as 35% of the DNA of the male gamete. A parallel study using Sciara ocellaris, a related species lacking L chromosomes, confirmed the presence of two X chromosomes in the sperm of this species.

Expression of HSP60 homologue in sheep blowfly Lucilia cuprina during development and heat stress

Among the evolutionarily most conserved molecular chaperones, members of HSP60 gene family (chaperonins) are assigned with different cellular functions, including significant role in proper folding of nascent polypeptides. We have made an attempt to specifically study the pattern of expression of HSP62, a member of HSP60 family, during development and heat stress in the sheep blowfly Lucilia cuprina, a pest of agricultural livestock. Studies have indicated constitutive expression of HSP62 in most of the larval and adult tissues. However, its level enhanced significantly upon heat shock in some tissues pointing its tentative role in tissue-specific thermoprotection. In situ localization of HSP60 by immunostaining or immunofluorescence in larval or adult tissues, using monoclonal anti-HSP60 antibodies, revealed its presence to be generally cytoplasmic. However, in certain polytene cell types, such as larval salivary glands, larval fat body or larval or adult Malpighian tubules, it appeared nuclear and cytoplasmic both. During oogenesis, HSP62 expression was found dynamic indicating its possible role in oocyte development. The differential levels of expression of HSP62 in different larval and adult tissues indicate their cell type specific requirement and thus, their transcription may be regulated in a tissue-specific manner. The present observations provide strong support to the role of HSP60 protein in thermotolerance, cell or tissue growth and differentiation.

Introgressive hybridization and evolution of a novel protein phenotype: Glue protein profiles in thenasuta-albomicans complex ofDrosophila

Glue proteins are tissue-specific proteins synthesized by larval salivary gland cells of Drosophila. In Drosophila nasuta nasuta and D. n. albomicans of the nasuta subgroup, the genes that encode the major glue protein fractions are X-linked. In the present study, these X-linked markers have been employed to trace the pattern of introgression of D. n. nasuta and D. n. albomicans genomes with respect to the major glue protein fractions in their interracial hybrids, called cytoraces. These cytoraces have inherited the chromosomes of both parents and have been maintained in the laboratory for over 400-550 generations. The analysis has revealed that cytoraces with D. n. albomicans X chromosome show either D. n. nasuta pattern or a completely novel pattern of glue protein fractions. Further, quantitative analysis also shows lack of correlation between the chromosomal pattern of inheritance and overall quantity of the major glue protein fractions in the cytoraces. Thus, in cytoraces the parental chromosomes are not just differentially represented but there is evidence for introgression even at the gene level.

Larval salivary glands are a source of primer and releaser pheromone in honey bee (Apis mellifera L.)

A brood pheromone identified in honeybee larvae has primer and releaser pheromone effects on adult bees. Using gas chromatography-mass spectrometry (GC-MS) to evaluate fatty acid esters--the pheromonal compounds--in different parts of the larvae, we have localized the source of the esters as the larval salivary glands. A histochemical study describes the glands and confirms the presence of lipids in the glands. Epithelial cells of the gland likely secrete the fatty acids into the lumen of the gland. These results demonstrate the salivary glands to be a reservoir of esters, components of brood pheromone, in honeybee larvae.

The Drosophila melanogaster Apaf-1 homologue ARK is required for most, but not all, programmed cell death

The Apaf-1 protein is essential for cytochrome c–mediated caspase-9 activation in the intrinsic mammalian pathway of apoptosis. Although Apaf-1 is the only known mammalian homologue of the Caenorhabditis elegans CED-4 protein, the deficiency of apaf-1 in cells or in mice results in a limited cell survival phenotype, suggesting that alternative mechanisms of caspase activation and apoptosis exist in mammals. In Drosophila melanogaster, the only Apaf-1/CED-4 homologue, ARK, is required for the activation of the caspase-9/CED-3–like caspase DRONC. Using specific mutants that are deficient for ark function, we demonstrate that ARK is essential for most programmed cell death (PCD) during D. melanogaster development, as well as for radiation-induced apoptosis. ark mutant embryos have extra cells, and tissues such as brain lobes and wing discs are enlarged. These tissues from ark mutant larvae lack detectable PCD. During metamorphosis, larval salivary gland removal was severely delayed in ark mutants. However, PCD occurred normally in the larval midgut, suggesting that ARK-independent cell death pathways also exist in D. melanogaster.

Exposure to low-dose X-rays promotes peculiar autophagic cell death in Drosophila melanogaster, an effect that can be regulated by the inducible expression of Hml dsRNA

We previously reported that to induce an early emergence effect with low-dose X-irradiation in Drosophila, exposure during the prepupae stage is necessary. The present study examined the mechanism by which low-dose radiation rapidly eliminates larval cells and activates the formation of the imaginal discs during metamorphosis. Upon exposure to 0.5 Gy X-rays at 2 h after puparium formation (APF), the larval salivary glands swelled and were surrounded by remarkably thick structures containing an acid phosphatase (Acph) enzyme, implicating a peculiar autophagic cell death. TUNEL staining revealed the presence of DNA fragmentations compared with cells from sham controls which remained unchanged until 12 h APF. Additionally, the salivary glands of exposed flies were completely destroyed by 10 h APF. Furthermore, exposure to 0.5 Gy X-rays also facilitated the activity of the engulfment function of dendritic cells (DCs); they were generated in the larval salivary glands, engulfed the cell corpses and finally moved to the fat body. Data from an experiment demonstrating the inducible expression of Hml double-stranded RNA (dsRNA) indicate that a slow rate of engulfment of larval cells results in a longer time to emergence. Thus, the animals subjected to low-dose X-rays activated autophagic processes, resulting in significantly faster adult eclosion.

Spotted-dick, a zinc-finger protein of Drosophila required for expression of Orc4 and S phase

The highly condensed chromosomes and chromosome breaks in mitotic cells of a Drosophila mutant, spotted-dick/pita, are the consequence of defects in DNA replication. Reduction of levels of Spotted-dick protein, by either RNAi or mutation, leads to the accumulation of cells that have DNA content intermediate to 2N and 4N in proliferating tissues and also compromises endoreduplication in larval salivary glands. The Spotted-dick Zinc-finger protein is present in the nuclei of cells committed to proliferation but necessary in cells undertaking S phase. We show that Spotted-dick/Pita functions as a transcription factor and that, in cultured S2 cells, it is an activator of expression of some 30 genes that include the Orc4 gene, required for initiation of DNA replication. Chromatin immunoprecipitation indicates that it associates with the genes that it activates in S2 cells together with other sites that could represent genes activated in other tissues. We discuss the role of Spotted-dick in the coordination of cellular growth and DNA replication.

Chromosomal Localization of Two Antihemostatic Salivary Factors in Simulium vittatum (Diptera: Simuliidae)

The chromosomal locations of two genes encoding the salivary protein products Simulidin (SVAT) and Simulium vittatum erythyma protein (SVEP) were identified using high-resolution cytogenetic mapping. Chromosomal subsection levels were determined from larval and adult salivary gland polytene chromosomes and adult Malphigian tubule chromosomes. Syntenic relationships occurred for both loci from a wild population of Simulium vittatum IIIL-1 collected in central Alabama and the colonized IS-7 S. vittatum sibling. cSVAT mapped to the short arm of chromosomes III, IIIS-72a4.5 and cSVEP mapped to the long arm of chromosome III, IIIL-96b1. cSVAT sits proximal to a common IIIS-2 paracentric inversion, which occurs predominately as the standard sequence in the IIIL-1 sibling and as the inverted sequence in the IS-7 sibling. cSVEP sits close to the differentiated X chromosomes in the IIIL-1 sibling.