Dissection and Staining of Drosophila Larval Ovaries

Many organs depend on stem cells for their development during embryogenesis and for maintenance or repair during adult life. Understanding how stem cells form, and how they interact with their environment is therefore crucial for understanding development, homeostasis and disease. The ovary of the fruit fly Drosophila melanogaster has served as an influential model for the interaction of germ line stem cells (GSCs) with their somatic support cells (niche) ^{1, 2}. The known location of the niche and the GSCs, coupled to the ability to genetically manipulate them, has allowed researchers to elucidate a variety of interactions between stem cells and their niches ^3-12.Despite the wealth of information about mechanisms controlling GSC maintenance and differentiation, relatively little is known about how GSCs and their somatic niches form during development. About 18 somatic niches, whose cellular components include terminal filament and cap cells (Figure 1), form during the third larval instar ^13-17. GSCs originate from primordial germ cells (PGCs). PGCs proliferate at early larval stages, but following the formation of the niche a subgroup of PGCs becomes GSCs ^{7, 16, 18, 19}. Together, the somatic niche cells and the GSCs make a functional unit that produces eggs throughout the lifetime of the organism.Many questions regarding the formation of the GSC unit remain unanswered. Processes such as coordination between precursor cells for niches and stem cell precursors, or the generation of asymmetry within PGCs as they become GSCs, can best be studied in the larva. However, a methodical study of larval ovary development is physically challenging. First, larval ovaries are small. Even at late larval stages they are only 100μm across. In addition, the ovaries are transparent and are embedded in a white fat body. Here we describe a step-by-step protocol for isolating ovaries from late third instar (LL3) Drosophila larvae, followed by staining with fluorescent antibodies. We offer some technical solutions to problems such as locating the ovaries, staining and washing tissues that do not sink, and making sure that antibodies penetrate into the tissue. This protocol can be applied to earlier larval stages and to larval testes as well.Download video file.^{(47M, mov)}     

One year of pomegranate juice intake decreases oxidative stress, inflammation, and incidence of infections in hemodialysis patients: a randomized placebo-controlled trial

Increased systemic inflammation and oxidative stress are well established as nontraditional key players in the pathogenesis of atherosclerosis and are also involved in the innate immunity dysregulation in hemodialysis (HD) patients. The study aim was to investigate the effect of 1-year intake of pomegranate juice, an antioxidant source, on oxidative stress, inflammation, and long-term clinical outcomes. A randomized placebo controlled double-blind trial was designed, enrolling 101 chronic HD patients to receive during each dialysis 100 cc of pomegranate juice, or matching placebo, three times a week for 1 year. The primary endpoints were levels of oxidative stress and inflammation biomarkers. Secondary endpoints were hospitalization due to infections and the progression of atherosclerotic process based on a composite of variables of the carotid arteries: intima media thickness (IMT), number, and structure of plaques. Pomegranate juice intake yielded a significant time response reduction in polymorphonuclear leukocyte priming, protein oxidation, lipid oxidation, and inflammation biomarkers levels. These beneficial effects were abolished 3 months postintervention. Pomegranate juice intake resulted in a significantly lower incidence rate of the second hospitalization due to infections. Furthermore, 25% of the patients in the pomegranate juice group had improvement and only 5% progression in the atherosclerotic process, while more than 50% of patients in the placebo group showed progression and none showed any improvement. Prolonged pomegranate juice intake improves nontraditional CV risk factors, attenuates the progression of the atherosclerotic process, strengthens the innate immunity, and thus reduces morbidity among HD patients.     

Addition of endothelial progenitor cells to renal revascularization restores medullary tubular oxygen consumption in swine renal artery stenosis

Renal artery stenosis (RAS) promotes microvascular rarefaction and fibrogenesis, which may eventuate in irreversible kidney injury. We have shown that percutaneous transluminal renal angioplasty (PTRA) or endothelial progenitor cells (EPC) improve renal cortical hemodynamics and function in the poststenotic kidney. The renal medulla is particularly sensitive to hypoxia, yet little is known about reversibility of medullary injury on restoration of renal blood flow. This study was designed to test the hypothesis that PTRA, with or without adjunct EPC delivery to the stenotic kidney, may improve medullary remodeling and tubular function. RAS was induced in 21 pigs using implantation of irritant coils, while another group served as normal controls (n = 7 each). Two RAS groups were then treated 6 wk later with PTRA or both PTRA and EPC. Four weeks later, medullary hemodynamics, microvascular architecture, and oxygen-dependent tubular function of the stenotic kidneys were examined using multidetector computed tomography, microcomputed tomography, and blood oxygenation level-dependent MRI, respectively. Medullary protein expression of vascular endothelial growth factor, endothelial nitric oxide synthase, hypoxia-inducible factor-1α, and NAD(P)H oxidase p47 were determined. All RAS groups showed decreased medullary vascular density and blood flow. However, in RAS+PTRA+EPC animals, EPC were engrafted in tubular structures, oxygen-dependent tubular function was normalized, and fibrosis attenuated, despite elevated expression of hypoxia-inducible factor-1α and sustained downregulation of vascular endothelial growth factor. In conclusion, EPC delivery, in addition to PTRA, restores medullary oxygen-dependent tubular function, despite impaired medullary blood and oxygen supply. These results support further development of cell-based therapy as an adjunct to revascularization of RAS.     

A triumvirate at the testis tip: sharing the power of stem cell control

Much attention has been given to the role of the niche in controlling stem cell maintenance and differentiation. However, cells other than niche cells might direct stem cell behavior. Evidence from the Drosophila reproductive system suggests that this is the case.     

FLP/FRT-mediated restoration of normal phenotypes and clonal sectors formation in rolC transgenic tobacco

Site-specific recombination systems have been shown to excise transgene DNA sequences positioned between their cognate target sites, and thus be used to generate clonal sectors in transgenic plants. Here we characterized clonal sectors derived from genetic reversion of rolC (A. rhizogenes) – induced vegetative and reproductive phenotypes, mediated by FLP recombinase from S. cerevisiae, in tobacco. The constitutive expression of rolC induces pleiotropic effects including reduced apical dominance and plant height, lanceolate and pale green leaves and small, male-sterile flowers. Two transgenic male-sterile tobacco lines (N. tabacum, Samsun NN) expressing a 35sP-rolC gene construct flanked by two FRT (FLP recombinase target) sites, were cross-pollinated with pollen from a constitutive 35sP-FLP expressing line. Three main phenotypes were generated in result of recombinase-mediated excision of the 35sP-rolC locus in the F₁ (FLP×FRT-35sP-rolC-FRT) hybrid progenies: (a) restoration of male fertility, associated with reversion to normal leaf phenotypes prior to flower bud formation, (b) development of normal and fertile lateral shoot sectors on the background of rolC-type plants, (c) restoration of partially fertile flowers, associated with display of peripheral normal leaf sectors surrounding rolC-type inner-leaf tissues, consistent with periclinal chimeras. These results, supported by DNA molecular analysis, indicate that site-specific recombination might be used as a relatively efficient tool for generation of transgenic periclinal chimeric plants.     

The tomato <Emphasis Type="Italic">dark green</Emphasis> mutation is a novel allele of the tomato homolog of the <Emphasis Type="Italic">DEETIOLATED1</Emphasis> gene

A comprehensive, multi-generation, allele test, carried out in this study, suggests that the tomato mutations dark-green (dg) and high pigment 2(j) (hp-2(j)) are allelic. The hp-2(j) mutant is caused by a mutation in the tomato homolog of the DEETIOLATED1 (DET1) gene, involved in the signal transduction cascade of light perception and morphogenesis. This suggestion is in agreement with the exaggerated photomorphogenic de-etiolation response of homozygous dg mutants grown under modulated light conditions. Sequence analysis of the DET1 gene was carried out in dg mutants representing two different lines, and revealed a single A-to-T base transversion in the second exon of the DET1 gene in comparison with the normal wild-type sequence. This transversion results in a conserved Asparagine(34)-to-Isoleucine(34) amino-acid substitution, and eliminates a recognition site for the AclI restriction endonuclease, present in the wild-type and in the other currently known tomato mutants at the DET1 locus. This polymorphism was used to develop a PCR-based DNA marker, which enables an early genotypic selection for breeding lycopene-rich tomatoes. Using this marker and sequence analysis we demonstrate that an identical base transversion also exists in dg mutants of the cultivar Manapal, in which the natural dg mutation was originally discovered. A linkage analysis, carried out in a F(2) population, shows a very strong linkage association between the DET1 locus of dg mutant plants and the photomorphogenic response of the seedlings, measured as hypocotyl length (12 < LOD Score < 13, R(2) = 51.1%). The results presented in this study strongly support the hypothesis that the tomato dg mutation is a novel allele of the tomato homolog of the DET1 gene.     

Injection of immature dendritic cells into adjuvant-treated skin obviates the need for ex vivo maturation

A key and limiting step in the process of generating human monocyte-derived dendritic cells (DC) for clinical applications is maturation. In the setting of immunotherapy, DC are matured ex vivo by culturing them with various agents that mimic the conditions encountered at a site of inflammation. This study examined whether the ex vivo DC maturation step could be replaced by maturing DC in situ by injecting immature DC into sites pre-exposed to agents that induce a microenvironment conducive to in situ maturation of the injected DC. The hypothesis was that recapitulation of the physiological conditions occurring during pathogen infection would lead to optimal conditions for DC maturation, migration, and function. Murine immature DC injected into adjuvant (Adjuprime, poly-arginine, or Imiquimod)-pretreated skin exhibited lymph node migratory capacity comparable to and immunostimulatory capacity equal to or exceeding that of ex vivo matured DC. Acquisition of migratory capacity did not always correlate with enhanced immunostimulatory capacity. Immunostimulatory capacity was not enhanced when mature DC were injected into adjuvant-pretreated sites and remained below that seen with immature DC matured in situ. Immature DC injected into adjuvant-pretreated sites were more effective than mature DC in stimulating antitumor immunity in mice. (111)Indium-labeled human monocyte-derived immature DC injected into adjuvant (Imiquimod)-pretreated sites in cancer patients acquired lymph node migratory capacity comparable to ex vivo matured DC. This study shows that in situ maturation offers a simpler and potentially superior method to generate potent immunostimulatory DC for clinical immunotherapy.     

Requirement of the MRN complex for ATM activation by DNA damage

The ATM protein kinase is a primary activator of the cellular response to DNA double-strand breaks (DSBs). In response to DSBs, ATM is activated and phosphorylates key players in various branches of the DNA damage response network. ATM deficiency causes the genetic disorder ataxia-telangiectasia (A-T), characterized by cerebellar degeneration, immunodeficiency, radiation sensitivity, chromosomal instability and cancer predisposition. The MRN complex, whose core contains the Mre11, Rad50 and Nbs1 proteins, is involved in the initial processing of DSBs. Hypomorphic mutations in the NBS1 and MRE11 genes lead to two other genomic instability disorders: the Nijmegen breakage syndrome (NBS) and A-T like disease (A-TLD), respectively. The order in which ATM and MRN act in the early phase of the DSB response is unclear. Here we show that functional MRN is required for ATM activation, and consequently for timely activation of ATM-mediated pathways. Collectively, these and previous results assign to components of the MRN complex roles upstream and downstream of ATM in the DNA damage response pathway and explain the clinical resemblance between A-T and A-TLD.     

c-Myc oncoprotein: cell cycle-related events and new therapeutic challenges in cancer and cardiovascular diseases

Advanced stages of both cancer and atherosclerosis are characterized by a local increase in tissue mass that may be hard to control. This increase in tissue mass can be attributed to oxidation-sensitive modification of cell cycle-related events, including cellular proliferation, differentiation, and apoptosis, which could be secondary to alteration in the activity of tumor suppressor gene and oncogene products. The oncogene c-Myc has classically been considered to be involved in carcinogenesis and has more recently been implicated in both endothelial dysfunction and atherogenesis as well. Consequently, inhibition of c-Myc-dependent signaling has become a novel therapeutic opportunity and challenge in atherosclerosis and other cardiovascular diseases. Antioxidant strategies, RNA synthesis inhibitors such as mithramycin, and gene therapeutic approaches with antisense oligonucleotides against c-Myc are some of the promising strategies. In general, the increased biologic understanding of the participation of cell cycle events and targeting these events may enable to attenuate or prevent some of the complications of vascular and neoplastic diseases.